U.S. patent number 6,403,675 [Application Number 09/070,204] was granted by the patent office on 2002-06-11 for biodegradable compositions comprising poly(cycloaliphatic phosphoester) compounds, articles, and methods for using the same.
This patent grant is currently assigned to Guilford Pharmaceuticals, Inc., Johns Hopkins University. Invention is credited to Wenbin Dang, James P. English, Irina Kadiyala, Kam W. Leong, Hai-quan Mao, Zhong Zhao.
United States Patent |
6,403,675 |
Dang , et al. |
June 11, 2002 |
Biodegradable compositions comprising poly(cycloaliphatic
phosphoester) compounds, articles, and methods for using the
same
Abstract
Biodegradable, flowable or flexible polymer compositions are
described comprising a polymer having the recurring monomeric units
shown in formula I: ##STR1## wherein: each of R and R' is
independently straight or branched alkylene, either unsubstituted
or substituted with one or more non-interfering substituents; L is
a divalent cycloaliphatic group; R" is selected from the group
consisting of H, alkyl, alkoxy, aryl, aryloxy, heterocyclic or
heterocycloxy; and n is 5 to 1,000, wherein said biodegradable
polymer is biocompatible before and upon biodegradation. In one
embodiment, one or more of R, R' or R" is a biologically active
substance. Amorphous compositions containing a biologically active
substance, in addition to the polymer, and methods for controllably
releasing biologically active substances using the compositions,
are also described.
Inventors: |
Dang; Wenbin (Ellicott City,
MD), Mao; Hai-quan (Towson, MD), Kadiyala; Irina
(Baltimore, MD), Leong; Kam W. (Ellicott City, MD), Zhao;
Zhong (Ellicott City, MD), English; James P. (Chelsea,
AL) |
Assignee: |
Guilford Pharmaceuticals, Inc.
(Baltimore, MD)
Johns Hopkins University (Baltimore, MD)
|
Family
ID: |
25284634 |
Appl.
No.: |
09/070,204 |
Filed: |
April 30, 1998 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
841345 |
Apr 30, 1997 |
|
|
|
|
Current U.S.
Class: |
523/113; 424/486;
528/400; 528/359; 424/78.08; 523/124; 524/610; 528/356; 523/111;
623/924 |
Current CPC
Class: |
A61K
47/34 (20130101); A61L 27/18 (20130101); A61L
31/06 (20130101); C08G 79/04 (20130101); A61L
27/18 (20130101); C08L 85/02 (20130101); A61L
31/06 (20130101); C08L 85/02 (20130101); Y10S
623/924 (20130101) |
Current International
Class: |
A61K
47/34 (20060101); A61L 27/18 (20060101); A61L
27/00 (20060101); A61L 31/04 (20060101); A61L
31/06 (20060101); C08G 79/00 (20060101); C08G
79/04 (20060101); A61K 031/80 (); C08G
079/04 () |
Field of
Search: |
;523/113,111 ;524/610
;528/354,356,400 ;424/486,78.08 ;623/1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Pretula, "High-molecular-weight Poly(alkylene phosphonate)s by
Condensation of Dialkylphosphonates with Diols", Die
Makromolekulare Chemie, 191:3, 371-80 (1990). .
Leong et al., "Polymeric Controlled Drug Delivery", Advanced Drug
Delivery Rev., 1:199-233 (1987). .
Langer, "New Methods of Drug Delivery", Science, 249:1527-33
(1990). .
Pulapura et al., "Trends in the Development of Bioresorbable
Polymers for Medical Applications", J. Biomaterials Appl., 6:1,
216-50 (1992). .
Bruin et al., "Biodegradable Lysine Diisocyanate-based
Poly(glycolide-co-.epsilon.-Caprolactone)-Urethane Network in
Artificial Skin", Biomaterials, 11:4, 291-95 (1990). .
Penczek et al., Handbook of Polymer Synthesis, Chapter 17:
"Phosphorus-Containing Polymers", (Hans R. Kricheldorf ed., 1992).
.
Sugiyama et al., "Preparation of Poly(phosphate ester)s Having
Bisphenol Moieties as Mesogenic Units in the Main Chain," Journal
of Polymer Science Part A: Polymer Chemistry Edition 32:11 (1994).
.
Wu, Xue Shen, "Synthesis and Properties of Biodegradable
Lactic/Glycolic Acid Polymers," Encyclopedic Handbook of
Biomaterials and Bioengineering, vol. 2: 1015 at 1016. .
Branham, Keith, "Synthesis and Characterization of Some
Organic-Inorganic Polymers," Dissertation, University of Alabama at
Birmingham, Abstract, 17-18 (1996). .
Lo, Hungnan, "Synthesis of Biodegradable Polymers and Porous Grafts
for Orthopedic Application," Thesis, Johns Hopkins University
(1995)..
|
Primary Examiner: Szekely; Peter
Attorney, Agent or Firm: Foley, Hoag & Eliot LLP
Parent Case Text
This application is a continuation-in-part application of U.S.
patent application Ser. No. 08/841,345, filed Apr. 30, 1997, now
abandoned, the contents of which are incorporated herein in their
entirety.
Claims
We claim:
1. A biodegradable, flowable or flexible polymer composition
comprising (a) a polymer having the recurring monomeric units shown
in formula I: ##STR12##
wherein:
each of R and R' is independently straight or branched aliphatic,
either unsubstituted or substituted with one or more
non-interfering substituents;
L is a divalent cycloaliphatic group, wherein the cyclic portion of
said cycloaliphatic group is not aromatic or heterocyclic in
nature;
R" is selected from the group consisting of H, alkyl, alkoxy, aryl,
aryloxy, heterocyclic or heterocycloxy; and
n is 5 to 1,000; and (b) at least one biologically active
substance,
wherein said biodegradable polymer composition is biocompatible
both before and upon biodegradation.
2. The polymer composition of claim 1 wherein each of R and R' is a
branched or straight chain alkylene group having from one to seven
carbon atoms.
3. The polymer composition of claim 1 wherein each of R and R' is a
methylene group or an ethylene group.
4. The polymer composition of claim 1 wherein R" is an alkyl group,
an alkoxy group, a phenyl group, a phenoxy group, or a
heterocycloxy group.
5. The polymer composition of claim 1 wherein R" is an alkoxy
group.
6. The polymer composition of claim 1 wherein n is 5 to 500.
7. The polymer composition of claim 1 wherein L is
cyclohexylene.
8. The polymer composition of claim 1 wherein said polymer
comprises additional biocompatible monomeric units or is blended
with other biocompatible polymers.
9. The polymer composition of claim 1 wherein said composition also
comprises at least one biologically active substance.
10. The polymer composition of claim 1 wherein said biologically
active substance is selected from the group consisting of peptides,
polypeptides, proteins, amino acids, polysaccharides, growth
factors, hormones, anti-angiogenesis factors, interferons or
cytokines, antigenic materials, and pro-drugs of these
substances.
11. The polymer composition of claim 1 wherein said biologically
active substance is a therapeutic drug or pro-drug.
12. The polymer composition of claim 11 wherein said drug is
selected from the group consisting of anti-neoplastic agents, local
anesthetics, antibiotics, anti-virals, anti-fungals,
anti-inflammatories, anticoagulants, antigenic materials suitable
for vaccine applications, and pro-drugs of these substances.
13. The polymer composition of claim 1 wherein said polymer
composition is non-toxic and results in minimal tissue irritation
when injected or is otherwise placed into intimate contact with
vasculated tissues.
14. A biodegradable, flowable or flexible polymer composition
comprising a polymer having the recurring monomeric units shown in
formula I: ##STR13##
wherein:
each of R and R' is independently straight or branched aliphatic,
either unsubstituted or substituted with one or more
non-interfering substituents;
L is a divalent cycloaliphatic group, wherein the cyclic portion of
said cycloaliphatic group is not aromatic or heterocyclic in
nature;
R" is selected from the group consisting of H, alkyl, alkoxy, aryl,
aryloxy, heterocyclic or heterocycloxy; and
n is 5 to 1,000,
or alternatively one or more of R, R' and R" is a biologically
active substance in a form capable of being released in a
physiological environment;
wherein said biodegradable polymer composition is biocompatible
both before and upon biodegradation.
15. The polymer composition of claim 14 wherein each of R and R' is
a branched or straight chain alkylene group having from one to
seven carbon atoms.
16. The polymer composition of claim 14 wherein each of R and R' is
a methylene group or an ethylene group.
17. The polymer composition of claim 14 wherein R" is an alkyl
group, an alkoxy group, a phenyl group, a phenoxy group, or a
heterocycloxy group.
18. The polymer composition of claim 14 wherein R" is an alkoxy
group.
19. The polymer composition of claim 14 wherein n is 5 to 500.
20. The polymer composition of claim 14 wherein L is
cyclohexylene.
21. The polymer composition of claim 14 wherein said polymer
comprises additional biocompatible monomeric units or is blended
with other biocompatible polymers.
22. The polymer composition of claim 14 wherein R" is a
biologically active substance.
23. The polymer composition of claim 14 wherein either R or R' is a
biologically active substance.
24. The polymer composition of claim 14 wherein said biologically
active substance is selected from the group consisting of peptides,
polypeptides, proteins, amino acids, polysaccharides, growth
factors, hormones, anti-angiogenesis factors, interferons or
cytokines, antigenic materials, and pro-drugs of these
substances.
25. The polymer composition of claim 14 wherein said biologically
active substance is a therapeutic drug or pro-drug.
26. The polymer composition of claim 25 wherein said drug is
selected from the group consisting of anti-neoplastic agents, local
anesthetics, antibiotics, anti-virals, anti-fungals,
anti-inflammatories, anticoagulants, antigenic materials suitable
for vaccine applications, and pro-drugs of these substances.
27. The polymer composition of claim 14 wherein said polymer
composition is non-toxic and results in minimal tissue irritation
when injected or is otherwise placed into intimate contact with
vasculated tissues.
28. The polymer composition of claim 11 wherein said drug is
selected from the group consisting of .beta.-adrenergic blocking
agents, anabolic agents, androgenic steroids, antacids,
anti-asthmatic agents, anti-allergenic materials, anti-arrhythmics,
anti-cholesterolemic and anti-lipid agents, anti-cholinergics and
sympathomimetics, anti-convulsants, anti-diarrheals, anti-emetics,
anti-hypertensive agents, anti-infective agents, anti-malarials,
anti-manic agents, anti-nauseants, anti-obesity agents,
anti-parkinsonian agents, anti-pyretic and analgesic agents,
anti-spasmodic agents, anti-thrombotic agents, anti-uricemic
agents, anti-anginal agents, antihistamines, anti-tussives,
appetite suppressants, benzophenanthridine alkaloids, biologicals,
cardioactive agents, cerebral dilators, coronary dilators,
decongestants, diuretics, diagnostic agents, erythropoietic agents,
estrogens, expectorants, gastrointestinal sedatives, humoral
agents, hyperglycemic agents, hypnotics, hypoglycemic agents, ion
exchange agents, laxatives, mineral supplements, miotics, mucolytic
agents, neuromuscular drugs, nutritional substances, peripheral
vasodilators, progestational agents, prostaglandins, psychic
energizers, psychotropics, sedatives, stimulants, thyroid and
anti-thyroid agents, tranquilizers, uterine relaxants, vitamins,
and pro-drugs of these substances.
29. The polymer composition of claim 25 wherein said drug is
selected from the group consisting of .beta.-adrenergic blocking
agents, anabolic agents, androgenic steroids, antacids,
anti-asthmatic agents, anti-allergenic materials, anti-arrhythmics,
anti-cholesterolemic and anti-lipid agents, anti-cholinergics and
sympathomimetics, anti-convulsants, anti-diarrheals, anti-emetics,
anti-hypertensive agents, anti-infective agents, anti-malarials,
anti-manic agents, anti-nauseants, anti-obesity agents,
anti-parkinsonian agents, anti-pyretic and analgesic agents,
anti-spasmodic agents, anti-thrombotic agents, anti-uricemic
agents, anti-anginal agents, antihistamines, anti-tussives,
appetite suppressants, benzophenanthridine alkaloids, biologicals,
cardioactive agents, cerebral dilators, coronary dilators,
decongestants, diuretics, diagnostic agents, erythropoietic agents,
estrogens, expectorants, gastrointestinal sedatives, humoral
agents, hyperglycemic agents, hypnotics, hypoglycemic agents, ion
exchange agents, laxatives, mineral supplements, miotics, mucolytic
agents, neuromuscular drugs, nutritional substances, peripheral
vasodilators, progestational agents, prostaglandins, psychic
energizers, psychotropics, sedatives, stimulants, thyroid and
anti-thyroid agents, tranquilizers, uterine relaxants, vitamins,
and pro-drugs of these substances.
30. The polymer composition of claim 1, wherein said biologically
active substance is lidocaine.
31. The polymer composition of claim 1, wherein said biologically
active substance is paclitaxel.
32. The polymer composition of claim 14, wherein said biologically
active substance is lidocaine.
33. The polymer composition of claim 14, wherein said biologically
active substance is paclitaxel.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to biodegradable poly(phosphoester)
compositions that degrade in vivo into non-toxic residues, in
particular those containing a cycloaliphatic structure in the
polymer backbone. The compositions of the invention are
particularly useful as flexible or flowable materials for
localized, controlled drug delivery systems.
2. Description of the Prior Art
Biocompatible polymeric materials have been used extensively in
therapeutic drug delivery and medical implant applications. If a
medical implant is intended for use as a drug delivery or other
controlled-release system, using a biodegradable polymeric carrier
is one effective means to deliver the therapeutic agent locally and
in a controlled fashion, see Langer et al., "Chemical and Physical
Structures of Polymers as Carriers for Controlled Release of
Bioactive Agents", J. Macro. Science, Rev. Macro. Chem. Phys.,
C23(1), 61-126 (1983). As a result, less total drug is required,
and toxic side effects can be minimized. Polymers have been used
for some time as carriers of therapeutic agents to effect a
localized and sustained release. See Leong et al., "Polymeric
Controlled Drug Delivery", Advanced Drug Delivery Rev., 1:199-233
(1987); Langer, "New Methods of Drug Delivery", Science,
249:1527-33 (1990) and Chien et al., Novel Drug Delivery Systems
(1982). Such delivery systems offer the potential of enhanced
therapeutic efficacy and reduced overall toxicity.
When a non-biodegradable polymer matrix is used, the steps leading
to release of the therapeutic agent are water diffusion into the
matrix, dissolution of the therapeutic agent, and diffusion of the
therapeutic agent out through the channels of the matrix. As a
consequence, the mean residence time of the therapeutic agent
existing in the soluble state is normally longer for a
non-biodegradable matrix than for a biodegradable matrix, for which
passage through the channels of the matrix, while it may occur, is
no longer required.
Since many pharmaceuticals have short half-lives, therapeutic
agents can decompose or become inactivated within the
non-biodegradable matrix before they are released. This issue is
particularly significant for many bio-macromolecules, e.g.,
proteins and smaller polypeptides, since these molecules are
generally hydrolytically unstable and have markedly low
permeabilities through most polymer matrices. In nonbiodegradable
matrices, many bio-macromolecules even begin to aggregate and
precipitate out of solution, blocking the very channels necessary
for diffusion out of the carrier matrix.
These problems are alleviated somewhat by using a biodegradable
rigid matrix that, in addition to some diffusional release,
primarily allows the controlled release of the therapeutic agent by
degradation of the solid polymer matrix. Examples of classes of
synthetic polymers that have been studied as possible solid
biodegradable materials include polyesters (Pitt et al.,
"Biodegradable Drug Delivery Systems Based on Aliphatic Polyesters:
Applications to Contraceptives and Narcotic Antagonists",
Controlled Release of Bioactive Materials, 19-44 (Richard Baker
ed., 1980); poly(amino acids) and pseudo-poly(amino acids)
(Pulapura et al. "Trends in the Development of Bioresorbable
Polymers for Medical Applications", J. Biomaterials Appl., 6:1,
216-50 (1992); polyurethanes (Bruin et al., "Biodegradable Lysine
Diisocyanate-based Poly(Glycolide-co-.epsilon.
Caprolactone)-Urethane Network in Artificial Skin", Biomaterials,
11:4, 291-95 (1990); polyorthoesters (Heller et al., "Release of
Norethindrone from Poly(Ortho Esters)", Polymer Engineering Sci.,
21:11, 727-31 (1981); and polyanhydrides (Leong et al.,
"Polyanhydrides for Controlled Release of Bioactive Agents",
Biomaterials 7:5, 364-71 (1986).
Polymers having phosphate linkages, called poly(phosphates),
poly(phosphonates) and poly(phosphites), are known. See Penczek et
al., Handbook of Polymer Synthesis, Chapter 17:
"Phosphorus-Containing Polymers", (Hans R. Kricheldorf ed., 1992).
The respective structures of these three classes of compounds, each
having a different side chain connected to the phosphorus atom, are
as follows: ##STR2##
The versatility of these polymers comes from the versatility of the
phosphorus atom, which is known for a multiplicity of reactions.
Its bonding can involve the 3porbitals or various 3s-3p hybrids;
spd hybrids are also possible because of the accessible dorbitals.
Thus, the physico-chemical properties of the poly(phosphoesters)
can be readily changed by varying either the R or R' group. The
biodegradability of the polymer is due primarily to the
physiologically labile phosphoester bond in the backbone of the
polymer. By manipulating the backbone or the side chain, a wide
range of biodegradation rates are attainable.
An additional feature of poly(phosphoesters) is the availability of
functional side groups. Because phosphorus can be pentavalent, drug
molecules or other biologically active substances can be chemically
linked to the polymer. For example, drugs with --O-carboxy groups
may be coupled to the phosphorus via a phosphoester bond, which is
hydrolyzable. See, Leong, U.S. Pat. Nos. 5,194,581 and 5,256,765.
The P--O--C group in the backbone also lowers the glass transition
temperature of the polymer and, importantly, confers solubility in
common organic solvents, which is desirable for easy
characterization and processing.
However, drug-delivery systems using most of the known
biodegradable polymers, including those of phosphoesters, have been
rigid materials. In such instances, the drug is incorporated into
the polymer, and the mixture is shaped into a certain form, such as
a cylinder, disc, or fiber for implantation.
However, proteins and other large biomolecules are still difficult
to deliver from rigid biodegradables because these larger molecules
are particularly unstable and are typically degraded along with the
solid polymeric matrix carrier. More specifically, when a polymer
begins to degrade following administration, a highly concentrated
microenvironment is created from the breakdown by-products of the
polymer as the polymer becomes ionized, protonated or hydrolyzed.
Proteins are easily denatured or degraded under these conditions
and then are useless for therapeutic purposes.
Further, in the process of preparing rigid drug delivery systems,
biologically active substances such as proteins are commonly
exposed to extreme stresses. Necessary manufacturing steps may
include excessive exposure to heat, pH extremes, large amounts of
organic solvents, cross-linking agents, freezing and drying.
Following manufacture or preparation, the drug delivery systems
must be stored for some extended period of time prior to
administration, and little information is available on the subject
of long term stability of proteins within solid biodegradable
delivery systems.
Rigid polymers can be inserted into the body with a syringe or
catheter in the form of small particles, such as microspheres or
microcapsules. However, because they are still solid particles,
they do not form the continuous and nearly homogeneous, monolithic
matrix that is sometimes needed for preferred release profiles.
In addition, microspheres or microcapsules prepared from these
polymers and containing biologically active substances to be
released into the body are sometimes difficult to produce on a
large scale. Most of the microencapsulation processes involve high
temperature and contact with organic solvents, steps that tend to
damage the bioactivity of proteins. Moreover, their storage often
presents problems and, upon injection, their granular nature can
cause blockages in injection devices and/or irritation of the soft
tissues into which the small particles are injected.
Dunn et al., U.S. Pat. Nos. 5,278,201; 5,278,202; and 5,340,849,
disclose a thermoplastic drug delivery system in which a solid,
linear-chain, biodegradable polymer or copolymer is dissolved in a
solvent to form a liquid solution. Once the polymer solution is
placed into the body where there is sufficient water, the solvent
dissipates or diffuses away from the polymer leaving it to
coagulate or solidify into a solid substance. However, the system
requires the presence of a solvent, and it is difficult to find an
organic solvent that is sufficiently non-toxic for acceptable
biocompatibility.
Thus, there exists a need for a composition and method for
providing a flexible or flowable biodegradable composition that can
be used in vivo to release a variety of different biologically
active substances, including hydrophobic drugs and even large and
bulky biomacromolecules, such as therapeutically useful proteins,
preferably without requiring the presence of significant amounts of
organic solvent. There is also a continuing need for biodegradable
polymer compositions that may provide controlled release in such a
way that trauma to the surrounding soft tissues can be
minimized.
Coover et al., U.S. Pat. No. 3,271,329, discloses organophosphorus
polymers prepared from dialkyl or diaryl hydrogen phosphites and
certain diol compounds, such as 1,4-cyclohexanedimethanol. See
column 1, lines 24-31. Vandenberg et al., U.S. Pat. No. 3,655,585,
discloses phosphorous polymers having at least one recurring unit
having the formula: ##STR3##
where R can be alkyl and Z can be alkylene such as cyclohexylene.
See column 1, lines 28-55. Herwig et al., U.S. Pat. No. 3,875,263,
discloses diphosphinic acid esters having a cyclic alkylene
portion, e.g., 1,4-methylene-cyclohexane. See column 1, lines 18-37
and column 2, line 13.
However, all of these patents suggest that such compounds and
polymeric compositions made from such compounds should be extruded
or molded to form articles or spun into fibers (Coover et al.);
used as additives for lubricating oils, gasoline, and synthetic
resins or other polymers (Vandenberg et al. and Herwig et al.); or
used as coating compounds (Herwig et al.). These compounds are
known by those of skill in the art primarily as conferring high
flame resistance and fire-proofing capabilities (Coover et al. and
Herwig et al.) or increased stability to oxidation and heat and
improved impact strength (Vandenberg et al.).
SUMMARY OF THE INVENTION
It has now been discovered that polymer compositions made with
poly(cycloaliphatic phosphoester) compounds provide conveniently
flexible or flowable carriers for even large and/or bulky
bio-macromolecules, including hydrophobic drugs and even large and
bulky bio-macromolecules, such as therapeutically useful proteins.
The biodegradable polymer composition of the invention comprises a
polymer having the recurring monomeric units shown in formula I:
##STR4##
wherein:
each of R and R' is independently straight or branched aliphatic,
either unsubstituted or substituted with one or more
non-interfering substituents;
L is a divalent cycloaliphatic group;
R" is selected from the group consisting of H, alkyl, alkoxy, aryl,
aryloxy, heterocyclic or heterocycloxy; and
n is 5 to 1,000
wherein the biodegradable polymer composition is biocompatible both
before and upon biodegradation. In a particularly preferred
embodiment, one or more of R, R' and R" is a biologically active
substance in a form capable of being released in a physiological
environment.
The invention also comprises a flexible article useful for
implantation, injection, or otherwise placed totally or partially
within the body, the article comprising a biodegradable, flowable
or flexible polymer composition comprising a polymer having the
recurring monomeric units shown in formula I where R, R', R", L and
n are as defined above.
In yet another embodiment of the invention, a method is provided
for the controlled release of a biologically active substance
comprising the steps of:
(a) combining the biologically active substance with a
biodegradable polymer having the recurring monomeric units shown in
formula I: ##STR5##
where R, R', L, R" and n are as defined above, to form an
implantable or injectable polymer composition; and
(b) placing the polymer composition formed in step (a) either
partially or totally within the body at a preselected site in vivo,
such that the polymer composition is in at least partial contact
with a biological fluid.
Because the compositions of the invention are preferably viscous,
flowable "gel-like" materials or flexible materials, they can be
used to deliver a wide variety of drugs, for example, from
hydrophobic drugs such as paclitaxel to large water-soluble
macromolecules such as proteins. Even when not flowable, the
compositions of the invention are still flexible and allow large
proteins to, at least partially, diffuse through the matrix prior
to the protein being degraded. The invention thus provides a
delivery system that is both convenient for use and capable of
delivering large bio-macromolecules in an effective manner.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the structure of P(trans-CHDM-HOP) as determined by
.sup.31 P-NMR and .sup.1 H-NMR.
FIG. 2 shows the chromatogram and molecular weight distribution for
P(cis-/trans-CHDM-HOP).
FIG. 3A graphically represents the active energy as a function of
frequency of P(trans- CHDM-HOP), and FIG. 3B shows temperature
dependence of the corresponding viscosity.
FIG. 4A shows HEK293 cells grown on a P(CHDM-HOP) surface after 72
hours of incubation, and FIG. 4B shows HEK293 cells grown on a TCPS
surface after 72 hours of incubation.
FIG. 5 graphically represents the effect of the side chain
structure on the in vitro degradation rate of three
poly(phosphoesters) of the invention in phosphate buffer
solution.
FIG. 6 shows the release curves of the bio-macromolecule FITC-BSA
from the polymer P(CHDM-HOP) at 33% loading.
FIG. 7 graphically represents the in vitro release kinetics of
FITC-BSA as a function of a loading levels of 30%, 10% and 1%.
FIG. 8 graphically represents the in vitro effect of side chain
structure on the protein release kinetics of FITC-BSA with a 10%
loading level.
FIG. 9 shows the release of low molecular weight drugs
(doxorubicin, cisplatin, and 5-fluorouracil) from P(CHDM-HOP).
FIG. 10 graphically represents the simultaneous release of
cisplatin and doxorubicin from a P(CHDM-HOP) matrix.
FIG. 11 graphically represents the cumulative percentage of
released IL-2 from the P(CHDM-HOP) matrix in phosphate butter as a
function of time.
FIG. 12 shows the calibration curves for the cumulative percentage
release of IL-2 from a P(CHDM-HOP) matrix in phosphate buffer.
FIG. 13 compares the pharmacokinetics of IL-2 administered as a
subcutaneous bolus or dispersed in a P(CHDM-HOP) matrix.
FIG. 14 shows the results of a histological examination of a
subcutaneous injection site in a Balb/c mouse.
FIG. 15 shows the distribution of tumor sizes in mice four weeks
after tumor implantation in an in vivo melanoma tumor model.
FIG. 16 shows the distribution of tumor sizes in mice six weeks
after tumor implantation in an in vivo melanoma tumor model.
FIG. 17 shows the percentage of survival as a function of time for
four different treatment groups in an in vivo melanoma tumor
model.
FIG. 18 shows the release curves of two polymer compositions of the
invention, one comprising the chemotherapeutic agent paclitaxel in
the polymer P(CHDM-EOP) and the other comprising paclitaxel in the
polymer P(CHDM-HOP).
FIG. 19 shows the in vitro release curves of lidocaine from three
different samples of P(CHDM-HOP)/lidocaine mixture.
FIG. 20A shows the cumulative amount of lidocaine released in vitro
as a function of incubation time, and FIG. 20B shows lidocaine
release as a function of the square root of time.
FIG. 21 plots the percentage of maximum nociceptive effect versus
time after in vivo injection of 25 mg of lidocaine in P(CHDM-HOP)
or in saline solution.
FIG. 22 plots the percentage of maximum motor function effect
versus time after injection of 25 mg of lidocaine in P(CHDM-HOP) or
in saline solution.
FIG. 23 shows the lidocaine concentration in plasma following
injection of 25 mg of lidocaine in saline solution, of 25 mg of
lidocaine in P(CHDM-HOP), and of 50 mg of lidocaine in
P(CHDM-HOP).
DETAILED DESCRIPTION OF THE INVENTION
Polymeric Compositions of the Invention
As used herein, the term "aliphatic" refers to a linear, branched
or cyclic alkane, alkene, or alkyne. Preferred linear or branched
aliphatic groups in the poly(cycloaliphatic phosphoester)
composition of the invention have from about 1 to 20 carbon atoms.
Preferred cycloaliphatic groups may have one or more sites of
unsaturation, i.e., double or triple bonds, but are not aromatic in
nature.
As used herein, the term "aryl" refers to an unsaturated cyclic
carbon compound with 4n+2 .pi. electrons. As used herein, the term
"heterocyclic" refers to a saturated or unsaturated ring compound
having one or more atoms other than carbon in the ring, for
example, nitrogen, oxygen or sulfur.
As used herein, the term "non-interfering substituent" means a
substituent that does react with the monomers; does not catalyze,
terminate or otherwise interfere with the polymerization reaction;
and does not react with the resulting polymer chain through intra-
or inter-molecular reactions.
The biodegradable and injectable polymer composition of the
invention comprises a polymer having the recurring monomeric units
shown in formula I: ##STR6##
wherein each of R and R' is independently straight or branched
aliphatic, either unsubstituted or substituted with one or more
non-interfering substituents. Each of R and R' can be any aliphatic
moiety so long as the moiety does not interfere undesirably with
the polymerization or biodegradation reactions of the polymer.
Preferably, R and R' have from about 1-20 carbon atoms. For
example, each of R and R' can be an alkylene group, such as
methylene, ethylene, 1,2-dimethylethylene, n-propylene,
isopropylene, 2-methylpropylene, 2,2-dimethylpropylene or
tert-butylene, n-pentylene, tert-pentylene, n-hexylene, n-heptylene
and the like; alkenylene, such as ethenylene, propenylene,
dodecenylene, and the like; alkynylene, such as propynylene,
hexynylene, octadecenynylene, and the like; an aliphatic group
substituted with a non-interfering substituent, for example,
hydroxy-, halogen- or nitrogen-substituted aliphatic group.
Preferably, however, each of R and R' is a branched or straight
chain alkylene group and, even more preferably, an alkylene group
having from 1 to 7 carbon atoms. Most preferably, R is a methylene
or ethylene group.
In one embodiment of the invention, either R, R', or both R and R',
can be a biologically active substance in a form capable of being
released in a physiological environment. When the biologically
active substance is part of the poly(phosphoester) backbone in this
way, it is released as the polymeric matrix formed by the
composition of the invention degrades.
Generally speaking, the biologically active substance of the
invention can vary widely with the purpose for the composition. The
term "biologically active substance" includes without limitation,
medicaments; vitamins; mineral supplements; substances used for the
treatment, prevention, diagnosis, cure or mitigation of a disease
or illness; substances which affect the structure or function of
the body; or pro-drugs, which become biologically active or more
active after they have been placed in a predetermined physiological
environment. The active substance(s) may be described as a single
entity or a combination of entities.
Non-limiting examples of broad categories of biologically active
substances include the following expanded therapeutic categories:
.beta.-adrenergic blocking agents, anabolic agents, androgenic
steroids, antacids, anti-asthmatic agents, anti-allergenic
materials, anti-cholesterolemic and anti-lipid agents,
anti-cholinergics and sympathomimetics, anti-coagulants,
anti-convulsants, anti-diarrheals, anti-emetics, anti-hypertensive
agents, anti-infective agents, anti-inflammatory agents such as
steroids, non-steroidal anti-inflammatory agents, anti-malarials,
anti-manic agents, anti-nauseants, anti-neoplastic agents,
anti-obesity agents, anti-parkinsonian agents, anti-pyretic and
analgesic agents, anti-spasmodic agents, anti-thrombotic agents,
anti-uricemic agents, anti-anginal agents, antihistamines,
anti-tussives, appetite suppressants, benzophenanthridine
alkaloids, biologicals, cardioactive agents, cerebral dilators,
coronary dilators, decongestants, diuretics, diagnostic agents,
erythropoietic agents, estrogens, expectorants, gastrointestinal
sedatives, humoral agents, hyperglycemic agents, hypnotics,
hypoglycemic agents, ion exchange resins, laxatives, mineral
supplements, miotics, imucolytic agents, neuromuscular drugs,
nutritional substances, peripheral vasodilators, progestational
agents, prostaglandins, psychic energizers, psychotropics,
sedatives, stimulants, thyroid and anti-thyroid agents,
tranquilizers, uterine relaxants, vitamins, antigenic materials,
and pro-drugs.
Specific examples of useful biologically active substances from the
above categories include: (a) anti-neoplastics such as androgen
inhibitors, antimetabolites, cytotoxic agents, and
immunomodulators; (b) anti-tussives such as dextromethorphan,
dextromethorphan hydrobromide, noscapine, carbetapentane citrate,
and chlorphedianol hydrochloride; (c) antihistamines such as
chlorpheniramine maleate, phenindamine tartrate, pyrilamine
maleate, doxylamine succinate, and phenyltoloxamine citrate; (d)
decongestants such as phenylephrine hydrochloride,
phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride,
and ephedrine; (e) various alkaloids such as codeine phosphate,
codeine sulfate, and morphine; (f) mineral supplements such as
potassium chloride, zinc chloride, calcium carbonate, magnesium
oxide, and other alkali metal and alkaline earth metal salts; (g)
ion exchange resins such as cholestryramine; (h) anti-arrhythmics
such as N-acetylprocainamide; (i) antipyretics and analgesics such
as acetaminophen, aspirin and ibuprofen; (j) appetite suppressants
such as phenyl-propanolamine hydrochloride or caffeine; (k)
expectorants such as guaifenesin; (l) antacids such as aluminum
hydroxide and magnesium hydroxide; (m) biologicals such as
peptides, polypeptides, proteins and amino acids, hormones,
interferons or cytokines and other bioactive peptidic compounds,
such as hGH, tPA, calcitonin, ANF, EPO and insulin; (n)
anti-infective agents such as anti-fungals, anti-virals,
antiseptics and antibiotics; and (m) desensitizing agents and
antigenic materials, such as those useful for vaccine
applications.
More specifically, non-limiting examples of useful biologically
active substances include the following therapeutic categories:
analgesics, such as nonsteroidal anti-inflammatory drugs, opiate
agonists and salicylates; antihistamines, such as H.sub.1 -blockers
and H.sub.2 -blockers; anti-infective agents, such as
antihelmintics, antianaerobics, antibiotics, aminoglycoside
antibiotics, antifungal antibiotics, cephalosporin antibiotics,
macrolide antibiotics, miscellaneous .beta.-lactam antibiotics,
penicillin antibiotics, quinolone antibiotics, sulfonamide
antibiotics, tetracycline antibiotics, antimycobacterials,
antituberculosis antimycobacterials, antiprotozoals, antimalarial
antiprotozoals, antiviral agents, anti-retroviral agents,
scabicides, and urinary anti-infectives; antineoplastic agents,
such as alkylating agents, nitrogen mustard alkylating agents,
nitrosourea alkylating agents, antimetabolites, purine analog
antimetabolites, pyrimidine analog antimetabolites, hormonal
antineoplastics, natural antineoplastics, antibiotic natural
antineoplastics, and vinca alkaloid natural antineoplastics;
autonomic agents, such as anticholinergics, antimuscarinic
anticholinergics, ergot alkaloids, parasympathomimetics,
cholinergic agonist parasympathomimetics, cholinesterase inhibitor
parasympathomimetics, sympatholytics, .alpha.-blocker
sympatholytics, .beta.-blocker sympatholytics, sympathomimetics,
and adrenergic agonist sympathomimetics; cardiovascular agents,
such as antianginals, .beta.-blocker antianginals, calcium-channel
blocker antianginals, nitrate antianginals, antiarrhythmics,
cardiac glycoside antiarrhythmics, class I antiarrhythmics, class
II antiarrhythmics, class III antiarrhythmics, class IV
antiarrhythmics, antihypertensive agents, .alpha.-blocker
antihypertensives, angiotensin-converting enzyme inhibitor (ACE
inhibitor) antihypertensives, .beta.-blocker antihypertensives,
calcium-channel blocker antihypertensives, central-acting
adrenergic antihypertensives, diuretic antihypertensive agents,
peripheral vasodilator antihypertensives, antilipemics, bile acid
sequestrant antilipemics, HMG-CoA reductase inhibitor antilipemics,
inotropes, cardiac glycoside inotropes, and thrombolytic agents;
dermatological agents, such as antihistamines, anti-inflammatory
agents, corticosteroid anti-inflammatory agents,
antipruritics/local anesthetics, topical anti-infectives,
antifungal topical anti-infectives, antiviral topical
anti-infectives, and topical antineoplastics; electrolytic and
renal agents, such as acidifying agents, alkalinizing agents,
diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics,
osmotic diuretics, potassium-sparing diuretics, thiazide diuretics,
electrolyte replacements, and uricosuric agents; enzymes, such as
pancreatic enzymes and thrombolytic enzymes; gastrointestinal
agents, such as antidiarrheals, antiemetics, gastrointestinal
anti-inflammatory agents, salicylate gastrointestinal
anti-inflammatory agents, antacid anti-ulcer agents, gastric
acid-pump inhibitor anti-ulcer agents, gastric mucosal anti-ulcer
agents, H.sub.2 -blocker anti-ulcer agents, cholelitholytic agents,
digestants, emetics, laxatives and stool softeners, and prokinetic
agents; general anesthetics, such as inhalation anesthetics,
halogenated inhalation anesthetics, intravenous anesthetics,
barbiturate intravenous anesthetics, benzodiazepine intravenous
anesthetics, and opiate agonist intravenous anesthetics;
hematological agents, such as antianemia agents, hematopoietic
antianemia agents, coagulation agents, anticoagulants, hemostatic
coagulation agents, platelet inhibitor coagulation agents,
thrombolytic enzyme coagulation agents, and plasma volume
expanders; hormones and hormone modifiers, such as abortifacients,
adrenal agents, corticosteroid adrenal agents, androgens,
antiandrogens, antidiabetic agents, sulfonylurea antidiabetic
agents, antihypoglycemic agents, oral contraceptives, progestin
contraceptives, estrogens, fertility agents, oxytocics, parathyroid
agents, pituitary hormones, progestins, antithyroid agents, thyroid
hormones, and tocolytics; immunobiologic agents, such as
immunoglobulins, immunosuppressives, toxoids, and vaccines; local
anesthetics, such as amide local anesthetics and ester local
anesthetics; musculoskeletal agents, such as anti-gout
anti-inflammatory agents, corticosteroid anti-inflammatory agents,
gold compound anti-inflammatory agents, immunosuppressive
anti-inflammatory agents, nonsteroidal anti-inflammatory drugs
(NSAIDs), salicylate anti-inflammatory agents, skeletal muscle
relaxants, neuromuscular blocker skeletal muscle relaxants, and
reverse neuromuscular blocker skeletal muscle relaxants;
neurological agents, such as anticonvulsants, barbiturate
anticonvulsants, benzodiazepine anticonvulsants, anti-migraine
agents, anti-parkinsonian agents, anti-vertigo agents, opiate
agonists, and opiate antagonists; ophthalmic agents, such as
anti-glaucoma agents, .beta.-blocker anti-glaucoma agents, miotic
anti-glaucoma agents, mydriatics, adrenergic agonist mydriatics,
antimuscarinic mydriatics, ophthalmic anesthetics, ophthalmic
anti-infectives, ophthalmic aminoglycoside anti-infectives,
ophthalmic macrolide anti-infectives, ophthalmic quinolone
anti-infectives, ophthalmic sulfonamide anti-infectives, ophthalmic
tetracycline anti-infectives, ophthalmic anti-inflammatory agents,
ophthalmic corticosteroid anti-inflammatory agents, and ophthalmic
nonsteroidal anti-inflammatory drugs (NSAIDs); psychotropic agents,
such as antidepressants, heterocyclic antidepressants, monoamine
oxidase inhibitors (MAOIs), selective serotonin re-uptake
inhibitors (SSRIs), tricyclic antidepressants, antimanics,
antipsychotics, phenothiazine antipsychotics, anxiolytics,
sedatives, and hypnotics, barbiturate sedatives and hypnotics,
benzodiazepine anxiolytics, sedatives, and hypnotics, and
psychostimulants; respiratory agents, such as antitussives,
bronchodilators, adrenergic agonist bronchodilators, antimuscarinic
bronchodilators, expectorants, mucolytic agents, respiratory
anti-inflammatory agents, and respiratory corticosteroid
anti-inflammatory agents; toxicology agents, such as antidotes,
heavy metal antagonists/chelating agents, substance abuse agents,
deterrent substance abuse agents, and withdrawal substance abuse
agents; minerals; and vitamins, such as vitamin A, vitamin B,
vitamin C, vitamin D, vitamin E, and vitamin K.
Preferred classes of useful biologically active substances from the
above categories include: (1) nonsteroidal anti-inflammatory drugs
(NSAIDs) analgesics, such as diclofenac, ibuprofen, ketoprofen, and
naproxen; (2) opiate agonist analgesics, such as codeine, fentanyl,
hydromorphone, and morphine; (3) salicylate analgesics, such as
aspirin (ASA) (enteric coated ASA); (4) H.sub.1 -blocker
antihistamines, such as clemastine and terfenadine; (5) H.sub.2
-blocker antihistamines, such as cimetidine, famotidine, nizadine,
and ranitidine; (6) anti-infective agents, such as mupirocin; (7)
antianaerobic anti-infectives, such as chloramphenicol and
clindamycin; (8) antifungal antibiotic anti-infectives, such as
amphotericin b, clotrimazole, fluconazole, and ketoconazole; (9)
macrolide antibiotic anti-infectives, such as azithromycin and
erythromycin; (10) miscellaneous .beta.-lactam antibiotic
anti-infectives, such as aztreonam and imipenem; (11) penicillin
antibiotic anti-infectives, such as nafcillin, oxacillin,
penicillin G, and penicillin V; (12) quinolone antibiotic
anti-infectives, such as ciprofloxacin and norfloxacin; (13)
tetracycline antibiotic anti-infectives, such as doxycycline,
minocycline, and tetracycline; (14) antituberculosis
antimycobacterial anti-infectives such as isoniazid (INH), and
rifampin; (15) antiprotozoal anti-infectives, such as atovaquone
and dapsone; (16) antimalarial antiprotozoal anti-infectives, such
as chloroquine and pyrimethamine; (17) anti-retroviral
anti-infectives, such as ritonavir and zidovudine; (18) antiviral
anti-infective agents, such as acyclovir, ganciclovir, interferon
alfa, and rimantadine; (19) alkylating antineoplastic agents, such
as carboplatin and cisplatin; (20) nitrosourea alkylating
antineoplastic agents, such as carmustine (BCNU); (21)
antimetabolite antineoplastic agents, such as methotrexate; (22)
pyrimidine analog antimetabolite antineoplastic agents, such as
fluorouracil (5-FU) and gemcitabine; (23) hormonal antineoplastics,
such as goserelin, leuprolide, and tamoxifen; (24) natural
antineoplastics, such as aldesleukin, interleukin-2, docetaxel,
etoposide (VP-16), interferon alfa, paclitaxel, and tretinoin
(ATRA); (25) antibiotic natural antineoplastics, such as bleomycin,
dactinomycin, daunorubicin, doxorubicin, and mitomycin; (26) vinca
alkaloid natural antineoplastics, such as vinblastine and
vincristine; (27) autonomic agents, such as nicotine; (28)
anticholinergic autonomic agents, such as benztropine and
trihexyphenidyl; (29) antimuscarinic anticholinergic autonomic
agents, such as atropine and oxybutynin; (30) ergot alkaloid
autonomic agents, such as bromocriptine; (31) cholinergic agonist
parasympathomimetics, such as pilocarpine; (32) cholinesterase
inhibitor parasympathomimetics, such as pyridostigmine; (33)
.alpha.-blocker sympatholytics, such as prazosin; (34)
.beta.-blocker sympatholytics, such as atenolol; (35) adrenergic
agonist sympathomimetics, such as albuterol and dobutamine; (36)
cardiovascular agents, such as aspirin (ASA) (enteric coated ASA);
(37) .beta.-blocker antianginals, such as atenolol and propranolol;
(38) calcium-channel blocker antianginals, such as nifedipine and
verapamil; (39) nitrate antianginals, such as isosorbide dinitrate
(ISDN); (40) cardiac glycoside antiarrhythmics, such as digoxin;
(41) class I antiarrhythmics, such as lidocaine, mexiletine,
phenytoin, procainamide, and quinidine; (42) class II
antiarrhythmics, such as atenolol, metoprolol, propranolol, and
timolol; (43) class III antiarrhythmics, such as amiodarone; (44)
class IV antiarrhythmics, such as diltiazem and verapamil; (45)
.alpha.-blocker antihypertensives, such as prazosin; (46)
angiotensin-converting enzyme inhibitor (ACE inhibitor)
antihypertensives, such as captopril and enalapril; (47)
.beta.-blocker antihypertensives, such as atenolol, metoprolol,
nadolol, and propanolol; (48) calcium-channel blocker
antihypertensive agents, such as diltiazem and nifedipine; (49)
central-acting adrenergic antihypertensives, such as clonidine and
methyldopa; (50) diurectic antihypertensive agents, such as
amiloride, furosemide, hydrochlorothiazide (HCTZ), and
spironolactone; (51) peripheral vasodilator antihypertensives, such
as hydralazine and minoxidil; (52) antilipemics, such as
gemfibrozil and probucol; (53) bile acid sequestrant antilipemics,
such as cholestyramine; (54) HMG-CoA reductase inhibitor
antilipemics, such as lovastatin and pravastatin; (55) inotropes,
such as amrinone, dobutamine, and dopamine; (56) cardiac glycoside
inotropes, such as digoxin; (57) thrombolytic agents, such as
alteplase (TPA), anistreplase, streptokinase, and urokinase; (58)
dermatological agents, such as colchicine, isotretinoin,
methotrexate, minoxidil, tretinoin (ATRA); (59) dermatological
corticosteroid anti-inflammatory agents, such as betamethasone and
dexamethasone; (60) antifungal topical anti-infectives, such as
amphotericin B, clotrimazole, miconazole, and nystatin; (61)
antiviral topical anti-infectives, such as acyclovir; (62) topical
antineoplastics, such as fluorouracil (5-FU); (63) electrolytic and
renal agents, such as lactulose; (64) loop diuretics, such as
furosemide; (65) potassium-sparing diuretics, such as triamterene;
(66) thiazide diuretics, such as hydrochlorothiazide (HCTZ); (67)
uricosuric agents, such as probenecid; (68) enzymes such as RNase
and DNase; (69) thrombolytic enzymes, such as alteplase,
anistreplase, streptokinase and urokinase; (70) antiemetics, such
as prochlorperazine; (71) salicylate gastrointestinal
anti-inflammatory agents, such as sulfasalazine; (72) gastric
acid-pump inhibitor anti-ulcer agents, such as omeprazole; (73)
H.sub.2 -blocker anti-ulcer agents, such as cimetidine, famotidine,
nizatidine, and ranitidine; (74) digestants, such as pancrelipase;
(75) prokinetic agents, such as erythromycin; (76) opiate agonist
intravenous anesthetics such as fentanyl; (77) hematopoietic
antianemia agents, such as erythropoietin, filgrastim (G-CSF), and
sargramostim (GM-CSF); (78) coagulation agents, such as
antihemophilic factors 1-10 (AHF 1-10); (79) anticoagulants, such
as warfarin; (80) thrombolytic enzyme coagulation agents, such as
alteplase, anistreplase, streptokinase and urokinase; (81) hormones
and hormone modifiers, such as bromocriptine; (82) abortifacients,
such as methotrexate; (83) antidiabetic agents, such as insulin;
(84) oral contraceptives, such as estrogen and progestin; (85)
progestin contraceptives, such as levonorgestrel and norgestrel;
(86) estrogens such as conjugated estrogens, diethylstilbestrol
(DES), estrogen (estradiol, estrone, and estropipate); (87)
fertility agents, such as clomiphene, human chorionic gonadotropin
(HCG), and menotropins; (88) parathyroid agents such as calcitonin;
(89) pituitary hormones, such as desmopressin, goserelin, oxytocin,
and vasopressin (ADH); (90) progestins, such as
medroxyprogesterone, norethindrone, and progesterone; (91) thyroid
hormones, such as levothyroxine; (92) immunobiologic agents, such
as interferon beta-lb and interferon gamma-1b; (93)
immunoglobulins, such as immune globulin IM, IMIG, IGIM and immune
globulin IV, IVIG, IGIV; (94) amide local anesthetics, such as
lidocaine; (95) ester local anesthetics, such as benzocaine and
procaine; (96) musculoskeletal corticosteroid anti-inflammatory
agents, such as beclomethasone, betamethasone, cortisone,
dexamethasone, hydrocortisone, and prednisone; (97) musculoskeletal
anti-inflammatory immunosuppressives, such as azathioprine,
cyclophosphamide, and methotrexate; (98) musculoskeletal
nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac,
ibuprofen, ketoprofen, ketorlac, and naproxen; (99) skeletal muscle
relaxants, such as baclofen, cyclobenzaprine, and diazepam; (100)
reverse neuromuscular blocker skeletal muscle relaxants, such as
pyridostigmine; (101) neurological agents, such as nimodipine,
riluzole, tacrine and ticlopidine; (102) anticonvulsants, such as
carbamazepine, gabapentin, lamotrigine, phenytoin, and valproic
acid; (103) barbiturate anticonvulsants, such as phenobarbital and
primidone; (104) benzodiazepine anticonvulsants, such as
clonazepam, diazepam, and lorazepam; (105) anti-parkinsonian
agents, such as bromocriptine, levodopa, carbidopa, and pergolide;
(106) anti-vertigo agents, such as meclizine; (107) opiate
agonists, such as codeine, fentanyl, hydromorphone, methadone, and
morphine; (108) opiate antagonists, such as naloxone; (109)
.beta.-blocker anti-glaucoma agents, such as timolol; (110) miotic
anti-glaucoma agents, such as pilocarpine; (111) ophthalmic
aminoglycoside anti-infectives, such as gentamicin, neomycin, and
tobramycin; (112) ophthalmic quinolone anti-infectives, such as
ciprofloxacin, norfloxacin, and ofloxacin; (113) ophthalmic
corticosteroid anti-inflammatory agents, such as dexamethasone and
prednisolone; (114) ophthalmic nonsteroidal anti-inflammatory drugs
(NSAIDs), such as diclofenac; (115) antipsychotics, such as
clozapine, haloperidol, and risperidone; (116) benzodiazepine
anxiolytics, sedatives and hypnotics, such as clonazepam, diazepam,
lorazepam, oxazepam, and prazepam; (117) psychostimulants, such as
methylphenidate and pemoline; (118) antitussives, such as codeine;
(119) bronchodilators, such as theophylline; (120) adrenergic
agonist bronchodilators, such as albuterol; (121) respiratory
corticosteroid anti-inflammatory agents, such as dexamethasone;
(122) antidotes, such as flumazenil and naloxone; (123) heavy metal
antagonists/chelating agents, such as penicillamine; (124)
deterrent substance abuse agents, such as disulfiram, naltrexone,
and nicotine; (125) withdrawal substance abuse agents, such as
bromocriptine; (126) minerals, such as iron, calcium, and
magnesium; (127) vitamin B compounds, such as cyanocobalamin
(vitamin B.sub.12) and niacin (vitamin B.sub.3); (128) vitamin C
compounds, such as ascorbic acid; and (129) vitamin D compounds,
such as calcitriol.
In addition to the foregoing, the following less common drugs may
also be used: chlorhexidine; estradiol cypionate in oil; estradiol
valerate in oil; flurbiprofen; flurbiprofen sodium; ivermectin;
levodopa; nafarelin; and somatropin.
Further, the following new drugs may also be used: recombinant
beta-glucan; bovine immunoglobulin concentrate; bovine superoxide
dismutase; the formulation comprising fluorouracil, epinephrine,
and bovine collagen; recombinant hirudin (r-Hir), HIV-1 immunogen;
human anti-TAC antibody; recombinant human growth hormone (r-hGH);
recombinant human hemoglobin (r-Hb); recombinant human mecasermin
(r-IGF-1); recombinant interferon beta-1a; lenograstim (G-CSF);
olanzapine; recombinant thyroid stimulating hormone (r-TSH); and
topotecan.
Further still, the following intravenous products may be used:
acyclovir sodium; aldesleukin; atenolol; bleomycin sulfate, human
calcitonin; salmon calcitonin; carboplatin; carmustine;
dactinomycin, daunorubicin HCl; docetaxel; doxorubicin HCl; epoetin
alfa; etoposide (VP-16); fluorouracil (5-FU); ganciclovir sodium;
gentamicin sulfate; interferon alfa; leuprolide acetate; meperidine
HCl; methadone HCl; methotrexate sodium; paclitaxel; ranitidine
HCl; vinblastin sulfate; and zidovudine (AZT).
Still further, the following listing of peptides, proteins, and
other large molecules may also be used, such as interleukins 1
through 18, including mutants and analogues; interferons .alpha.,
.beta., and .gamma.; luteinizing hormone releasing hormone (LHRH)
and analogues, gonadatropin releasing hormone (GnRH), transforming
growth factor-.beta. (TGF-.beta.); fibroblast growth factor (FGF);
tumor necrosis factor-.alpha. & .beta. (TNF-.alpha. &
.beta.); nerve growth factor (NGF); growth hormone releasing factor
(GHRF); epidermal growth factor (EGF); fibroblast growth factor
homologous factor (FGFHF); hepatocyte growth factor (HGF); insulin
growth factor (IGF); platelet-derived growth factor (PDGF);
invasion inhibiting factor-2 (IIF-2); bone morphogenetic proteins
1-7 (BMP 1-7); somatostatin; thymosin-.alpha.-1; .gamma.-globulin;
superoxide dismutase (SOD); and complement factors.
Alternatively, the biologically active substance may be nucleic
acids comprised of nucleotides linked together into polynucleotide
chains with backbones consisting of alternating series of pentose
sugars and phosphate residues. One way to avoid the complications
of developing cell-based systems for delivering genes to patients
in gene therapy is to deliver retroviral vectors directly to target
cells. For example, this technique has been used to infect
endothelial cells of blood vessel walls. The polymers and
compositions of the invention may be used for direct delivery of
such retroviral vectors and/or related genetic materials to other
sites in vivo, for example, to the lungs to treat ailments in the
lungs, such as cystic fibrosis, or to treat tumors in any localized
portion of the body.
Preferably, the biologically active substance is selected from the
group consisting of peptides, polypeptides, proteins, amino acids,
polysaccharides, growth factors, hormones, anti-angiogenesis
factors, interferons or cytokines, antigenic materials, and
pro-drugs. In a particularly preferred embodiment, the biologically
active substance is a therapeutic drug or pro-drug, most preferably
a drug selected from the group consisting of chemotherapeutic
agents and other anti-neoplastics such as paclitaxel, antibiotics,
anti-virals, anti-fungals, anti-inflammatories, and anticoagulants,
antigens useful for vaccine applications or corresponding
pro-drugs.
Various forms of the biologically active agents may be used. These
include, without limitation, such forms as uncharged molecules,
molecular complexes, salts, ethers, esters, amides, and the like,
which are biologically activated when implanted, injected or
otherwise placed into the body.
L in the polymer composition of the invention can be any divalent
cycloaliphatic group so long as it does not interfere with the
polymerization or biodegradation reactions of the polymer of the
composition. Specific examples of useful L groups include
unsubstituted and substituted cycloalkylene groups, such as
cyclopentylene, 2-methyl-cyclopentylene, cyclohexylene,
2-chlorocyclohexylene, and the like; cycloalkenylene groups, such
as cyclohexenylene; and cycloalkylene groups having fused or
bridged additional ring structures on one or more sides, such as
tetralinylene, decalinylene, and norpinanylene; or the like.
R" in the polymer composition of the invention is an alkyl, alkoxy,
aryl, aryloxy, heterocyclic or heterocycloxy residue. Examples of
useful alkyl R" groups include methyl, ethyl, n-propyl, i-propyl,
n-butyl, tert-butyl, --C.sub.8 H.sub.17, and the like groups; alkyl
substituted with a non-interfering substituent, such as a halogen
group; corresponding alkoxy groups; and alkyl that is conjugated
with a biologically active substance to form a pendant drug
delivery system.
When R" is alkyl or alkoxy, it preferably contains about 2 to about
20 carbon atoms, even more preferably about 6 to about 15 carbon
atoms. When R" is aryl or the corresponding aryloxy group, it
typically contains from about 5 to about 14 carbon atoms,
preferably about 5 to 12 carbon atoms and, optionally, can contain
one or more rings that are fused to each other. Examples of
particularly suitable aromatic groups include phenyl, phenoxy,
naphthyl, anthracenyl, phenanthrenyl and the like.
When R" is heterocyclic or heterocycloxy, it typically contains
from about 5 to 14 ring atoms, preferably from about 5 to 12 ring
atoms, and one or more heteroatoms. Examples of suitable
heterocyclic groups include furan, thiophene, pyrrole, isopyrrole,
3-isopyrrole, pyrazole, 2-isoimidazole, 1,2,3-triazole,
1,2,4-triazole, oxazole, thiazole, isothiazole, 1,2,3-oxadiazole,
1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole,
1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-dioxazole,
1,2,4-dioxazole, 1,3,2-dioxazole, 1,3,4-dioxazole,
1,2,5-oxatriazole, 1,3-oxathiole, 1,2-pyran, 1,4-pyran, 1,2-pyrone,
1,4-pyrone, 1,2-dioxin-, 1,3-dioxin, pyridine, N-alkyl pyridinium,
pyridazine, pyrimidine, pyrazine, 1,3,5-triazine, 1,2,4-triazine,
1,2,3-triazine, 1,2,4-oxazine, 1,3,2-oxazine, 1,3,5-oxazine,
1,4-oxazine, o-isoxazine, p-isoxazine, 1,2,5-oxathiazine,
1,2,6-oxathiazine, 1,4,2-oxadiazine, 1,3,5,2-oxadiazine, azepine,
oxepin, thiepin, 1,2,4-diazepine, indene, isoindene, benzofuran,
isobenzofuran, thionaphthene, isothionaphthene, indole, indolenine,
2-isobenzazole, 1,4-pyrindine, pyrando[3,4-b]-pyrrole, isoindazole,
indoxazine, benzoxazole, anthranil, 1,2-benzopyran,
1,2-benzopyrone, 1,4-benzopyrone, 2,1-benzopyrone, 2,3-benzopyrone,
quinoline, isoquinoline, 12,-benzodiazine, 1,3-benzodiazine,
naphthpyridine, pyrido[3,4-b]-pyridine, pyrido[3,2-b]-pyridine,
pyrido[4,3-b]pyridine, 1,3,2-benzoxazine, 1,4,2-benzoxazine,
2,3,1-benzoxazine, 3,1,4-benzoxazine, 1,2-benzisoxazine,
1,4-benzisoxazine, carbazole, xanthrene, acridine, purine, and the
like. Preferably, when R" is heterocyclic or heterocycloxy, it is
selected from the group consisting of furan, pyridine,
N-alkylpyridine, 1,2,3- and 1,2,4-triazoles, indene, anthracene and
purine rings.
In a particularly preferred embodiment, R" is an alkyl group, an
alkoxy group, a phenyl group, a phenoxy group, or a heterocycloxy
group and, even more preferably, an alkoxy group having from 1 to
10 carbon atoms. Most preferably, R" is an ethoxy or hexyloxy
group.
Alternatively, the side chain R" can be a biologically active
substance pendently attached to the polymer backbone, for example
by ionic or covalent bonding. In this pendant system, the
biologically active substance is released as the bond connecting R"
with the phosphorous atom is cleaved under physiological
conditions.
The number "n" can vary greatly depending on the biodegradability
and the release characteristics desired in the polymer, but
typically varies between about 5 and 1,000. Preferably, n is from
about 5 to about 500 and, most preferably, from about 5 to about
200.
The molecular weight of the polymer used in the composition of the
invention can vary widely, but must remain low enough for the
polymer to maintain its flowable or flexible state. For example,
weight-average molecular weights (Mw) typically vary from about
2,000 to about 400,000 daltons, preferably from about 2,000 to
about 200,000 daltons and, most preferably, from about 2,000 to
50,000 daltons. Number-average molecular weight (Mn) can also vary
widely, but generally fall in the range of about 1,000 to about
200,000 daltons, preferably from about 1,000 to about 100,000
daltons and, most preferably, from about 1,000 to about 25,000
daltons.
Biodegradable polymers differ from non-biodegradable polymers in
that they can be degraded during in vivo therapy. This generally
involves breaking down the polymer into its monomeric subunits. In
principle, the ultimate hydrolytic breakdown products of the
polymer used in the invention are a cycloaliphatic diol, an
aliphatic alcohol and phosphate. All of these degradation products
are potentially non-toxic. However, the intermediate oligomeric
products of the hydrolysis may have different properties. Thus, the
toxicology of a biodegradable polymer intended for injection or
placing totally or partially within the body, even one synthesized
from apparently innocuous monomeric structures, is typically
determined after one or more toxicity analyses.
There are many different ways of testing for toxicity and/or
biocompatibility known to those of ordinary skill in the art. A
typical in vitro toxicity assay, however, would be performed with
live carcinoma cells, such as GT3TKB tumor cells, in the following
manner:
Two hundred microliters of various concentrations of the degraded
polymer products are placed in 96-well tissue culture plates seeded
with human gastric carcinoma cells (GT3TKB) at 10.sup.4 /well
density. The degraded polymer products are incubated with the
GT3TKB cells for 48 hours. The results of the assay can be plotted
as % relative growth vs. concentration of degraded polymer in the
tissue-culture well.
Polymers for use in medical applications, such as drug delivery
systems, can also be evaluated by well-known in vivo
biocompatibility tests, such as by subcutaneous implantation or
injection in rats to confirm that the systems hydrolyze without
significant levels of irritation or inflammation at the insertion
site.
The biodegradable polymer used in the invention is preferably
sufficiently pure to be biocompatible itself and remains
biocompatible upon biodegradation. By "biocompatible", it is meant
that the biodegradation products or the polymer itself are
non-toxic and result in only minimal tissue irritation when
injected or placed into intimate contact with vasculated tissues.
The requirement for biocompatibility is more easily accomplished
because the presence of an organic solvent is not required in the
polymer composition of the invention.
However, the polymer used in the invention is preferably soluble in
one or more common organic solvents for ease of synthesis,
purification and handling. Common organic solvents include such
solvents as ethanol, chloroform, dichloromethane, acetone, ethyl
acetate, DMAC, N-methyl pyrrolidone, dimethylformamide, and
dimethylsulfoxide. The polymer is preferably soluble in at least
one of the above solvents.
The polymer of the invention can also comprise additional
biocompatible monomeric units so long as they do not interfere with
the biodegradable characteristics and the desirable flow
characteristics of the invention. Such additional monomeric units
may offer even greater flexibility in designing the precise release
profile desired for targeted drug delivery or the precise rate of
biodegradability desired for other applications. When such
additional monomeric units are used, however, they should be used
in small enough amounts to insure the production of a biodegradable
copolymer having the desired physical characteristics, such as
viscosity, flowability, flexibility or morphology.
Examples of such additional biocompatible monomers include the
recurring units found in other poly(phosphoesters), poly(lactides),
poly(glycolides), poly(caprolactones), poly~anhydrides)J
poly(amides), poly(urethanes), poly(esteramides),
poly(orthoesters), poly(dioxanones), poly(acetals), poly(ketals),
poly(carbonates), poly(orthocarbonates), poly(phosphazenes),
poly(hydroxybutyrates), poly(hydroxyvalerates), poly(alkylene
oxalates), poly(alkylene succinates), poly(malic acids), poly(amino
acids), poly(vinylpyrrolidone), poly(ethylene glycol),
poly(hydroxycellulose), chitin, chitosan, and copolymers,
terpolymers, or combinations or mixtures of the above
materials.
When additional monomeric units are used, those which have a lower
degree of crystallization and are more hydrophobic are preferred.
Especially preferred recurring units with the desired physical
characteristics are those derived from poly(lactides),
poly(caprolactones), and copolymers of these with glycolide, in
which there are more amorphous regions.
Synthesis of Poly(cycloaliphatic phosphoester) Polymers
The most common general reaction in preparing poly-(phosphates) is
a dehydrochlorination between a phosphorodichloridate and a diol
according to the following equation: ##STR7##
Most poly(phosphonates) are also obtained by condensation between
appropriately substituted dichlorides and diols.
Poly(phosphites) have been prepared from glycols in a two-step
condensation reaction. A 20% molar excess of a dimethylphosphite is
used to react with the glycol, followed by the removal of the
methoxyphosphonyl end groups in the oligomers by high
temperature.
An advantage of melt polycondensation is that it avoids the use of
solvents and large amounts of other additives, thus making
purification more straightforward. It can also provide polymers of
reasonably high molecular weight. Somewhat rigorous conditions,
however, are often required and can lead to chain acidolysis (or
hydrolysis if water is present). Unwanted, thermally-induced side
reactions, such as crosslinking reactions, can also occur if the
polymer backbone is susceptible to hydrogen atom abstraction or
oxidation with subsequent macroradical recombination.
To minimize these side reactions, the polymerization can also be
carried out in solution. Solution polycondensation requires that
both the prepolymer and the phosphorus component be soluble in a
common solvent. Typically, a chlorinated organic solvent is used,
such as chloroform, dichloromethane, or dichloroethane.
The solution polymerization is preferably run in the presence of
equimolar amounts of the reactants and a stoichiometric amount of
an acid acceptor, usually a tertiary amine such as pyridine or
triethylamine. The product is then typically isolated from the
solution by precipitation in a non-solvent and purified to remove
the hydrochloride salt by conventional techniques known to those of
ordinary skill in the art, such as by washing with an aqueous
acidic solution, e.g., dilute HCl.
Reaction times tend to be longer with solution polymerization than
with melt polymerization. However, because overall milder reaction
conditions can be used, side reactions are minimized, and more
sensitive functional groups can be incorporated into the polymer.
Moreover, attainment of undesirably high molecular weights is less
likely with solution polymerization.
Interfacial polycondensation can be used when high reaction rates
are desired. The mild conditions used minimize side reactions, and
there is no need for stoichiometric equivalence between the diol
and dichloridate starting materials as in solution methods.
However, hydrolysis of the acid chloride may occur in the alkaline
aqueous phase. Sensitive dichloridates that have some solubility in
water are generally subject to hydrolysis rather than
polymerization. Phase transfer catalysts, such as crown ethers or
tertiary ammonium chloride, can be used to bring the ionized diol
to the interface to facilitate the polycondensation reaction. The
yield and molecular weight of the resulting polymer after
interfacial polycondensation are affected by reaction time, molar
ratio of the monomers, volume ratio of the immiscible solvents, the
type of acid acceptor, and the type and concentration of the phase
transfer catalyst.
In a preferred embodiment of the invention, the biodegradable
polymer of formula I is made by a process comprising the step of
reacting a diol having the formula:
wherein R, R' and L are as defined above, with a
phosphorodihalidate of formula II: ##STR8##
where "halo" is Br, Cl or I, and R- is as defined above, to form
the polymer of formula I. The diol HO--R--L--R'--OH can be prepared
by standard procedures of chemistry, and many such compounds are
available on a commercial basis.
When either R or R' is a biologically active substance, the
biologically active substance is preferably itself a diol, for
example, a steroid such as estradiol. Alternatively, the
biologically active substance can be a diamino compound that is
reacted with the carboxyl group of a carboxylic acid to produce
terminal hydroxyl groups that can be used to form the
poly(phosphoester) structure.
The purpose of the polymerization reaction is to form a polymer
comprising (i) cycloaliphatic recurring units and (ii) phosphoester
recurring units. The result can be a homopolymer, a relatively
homogeneous copolymer, or a block copolymer that has a somewhat
heterogeneous microcrystalline structure. Any one of these three
embodiments is well-suited for use as a controlled release
medium.
The process used to make the polymers used in the invention can
take place at widely varying temperatures, depending upon whether a
solvent is used and, if so, which one; the molecular weight
desired; the solubility desired; the susceptibility of the
reactants to form side reactions; and the presence of a catalyst.
Preferably, however, the process takes place at a temperature
ranging from about 0 to about +235.degree. C. for melt conditions.
Somewhat lower temperatures, e.g., from about -50 to about
100.degree. C., may be possible with solution polymerization or
with the use of either a cationic or anionic catalyst.
The time required for the process can also vary widely, depending
upon the type of reaction being used, the molecular weight desired
and, in general, the need to use more or less rigorous conditions
for the reaction to proceed to the desired degree of completion.
Typically, however, the process takes place during a time between
about 30 minutes and 4 days.
While the process may be in bulk, in solution, by interfacial
polycondensation, or any other convenient method of polymerization,
preferably, the process takes place under solution conditions.
Particularly useful solvents include methylene chloride,
chloroform, tetrahydrofuran, dimethyl formamide, dimethyl
sulfoxide, toluene, or any of a wide variety of other inert organic
solvents.
Particularly when solution polymerization reaction is used, an acid
acceptor is advantageously present during the polymerization
reaction. A particularly suitable class of acid acceptor comprises
tertiary amines, such as pyridine, trimethylamine, triethylamine,
substituted anilines and substituted aminopyridines. The most
preferred acid acceptor is the substituted aminopyridine
4-dimethylaminopyridine ("DMAP").
The addition sequence for solution polymerization can vary
significantly depending upon the relative reactivities of the diol;
the phosphorodihalidate of formula II; the purity of these
reactants; the temperature at which the polymerization reaction is
preformed; the degree of agitation used in the polymerization
reaction; and the like. Preferably, however, the diol is combined
with a solvent and an acid acceptor, and then the
phosphorodihalidate is added slowly. For example, a solution of the
phosphorodihalidate in a solvent may be trickled in or added
dropwise to the chilled reaction mixture of diol, solvent and acid
acceptor, to control the rate of the polymerization reaction.
The polymer of formula I is isolated from the reaction mixture by
conventional techniques, such as by precipitating out, extraction
with an immiscible solvent, evaporation, filtration,
crystallization and the like. Typically, however, the polymer of
formula I is both isolated and purified by quenching a solution of
the polymer with a non-solvent or a partial solvent, such as
diethyl ether or petroleum ether.
Biodegradability and Release Characteristics
The polymer of formula I is usually characterized by a
biodegradation rate that is controlled at least in part as a
function of hydrolysis of the phosphoester bond of the polymer.
Other factors are also important. For example, the lifetime of a
biodegradable polymer in vivo also depends upon its molecular
weight, crystallinity, biostability, and the degree of
crosslinking. In general, the greater the molecular weight, the
higher the degree of crystallinity, and the greater the
biostability, the slower biodegradation will be. In addition, the
rate of degradation of the polymer can be further controlled by
choosing a side chain of differing lengths. Accordingly,
degradation times can very widely, preferably from less than a day
to several months.
Accordingly, the structure of the side chain can influence the
release behavior of compositions comprising a biologically active
substance. For example, it is expected that conversion of the
phosphate side chain to a more lipophilic, more hydrophobic or
bulky group would slow down the degradation process. Thus, release
is usually faster from polymer compositions with a small aliphatic
group side chain than with a bulky aromatic side chain. Moreover,
when R and/or R' in the backbone portion of formula I is itself a
biologically active substance, the release rate of the biologically
active substance in vivo is primarily governed by the rate of
biodegradation. When the biologically active substance to be
released is conjugated to the phosphorus side chain R" to form a
pendant drug delivery system, the release profile is governed to a
significant degree by the lability of the phosphorous-R" bond.
The mechanical properties of the polymer are also important with
respect to the flowability or flexibility of the composition
containing the polymer. For example, the glass transition
temperature is preferably low enough to keep the composition of the
invention flowable at body temperature. Even more preferably, the
glass transition temperature of the polymer used in the invention
is about 0 to about 37.degree. C. and, most preferably, from about
0 to about 25.degree. C.
Polymer Compositions
The polymer composition of the invention may be a flexible or
flowable material. By "flowable" is meant the ability to assume,
over time, the shape of the space containing it at body
temperature. This includes, for example, liquid compositions that
are capable of being sprayed into a site; injected with a manually
operated syringe fitted with, for example, a 23-gauge needle; or
delivered through a catheter.
Also included by the term "flowable", however, are highly viscous,
"gel-like" materials at room temperature that may be delivered to
the desired site by pouring, squeezing from a tube, or being
injected with any one of the commercially available power injection
devices that provide injection pressures greater than would be
exerted by manual means alone for highly viscous, but still
flowable, materials. When the polymer used is itself flowable, the
polymer composition of the invention, even when viscous, need not
include a biocompatible solvent to be flowable, although trace or
residual amounts of biocompatible solvents may still be present.
The viscosity of the polymer can be adjusted by the molecular
weight of the polymer, as well as by mixing the cis- and trans-
isomers of the cyclohexane dimethanol in the backbone of the
polymer.
Even without the presence of a biologically active substance, the
polymer composition of the invention can be used for a variety of
medical applications. For example, it can be injected to form,
after injection, a temporary biomechanical barrier to coat or
encapsulate internal organs or tissues, such as the barriers used
to prevent adhesions after abdominal surgery. The polymer
composition of the invention can also be used to produce bone waxes
and fillers for repairing injuries to bone or connective tissue,
temporary internal "bandages" to prevent further internal injury or
promote internal wound healing, or coatings for solid implantable
devices.
The biodegradable composition can even be injected subdermally to
build up tissue or to fill in defects. The injected polymer
composition will slowly biodegrade within the body and allow
natural tissue to grow and replace the polymer matrix as it
disappears. Thus, when the material is injected into a soft-tissue
defect, it will fill that defect and provide a scaffold for natural
collagen tissue to grow. This collagen tissue will gradually
replace the biodegradable polymer. However, preferably, the polymer
composition of the invention does comprise a biologically active
substance and provides controllable and effective release of the
biologically active substance over time, even in the case of large
bio-macromolecules. Thus, in a preferred embodiment, the
biodegradable polymer composition comprises both:
(a) at least one biologically active substance and
(b) the polymer having the recurring monomeric units shown in
formula I where R, R' , L, R" and n are as defined above.
The biologically active substances are used in amounts that are
therapeutically effective, which varies widely depending largely on
the particular biologically active substance being used. The amount
of biologically active substance incorporated into the composition
also depends upon the desired release profile, the concentration of
the substance required for a biological effect, and the length of
time that the biologically active substance has to be released for
treatment. Preferably, the biologically active substance can be
easily blended with the polymer matrix of the invention at
different loading levels, at room temperature and without the need
for an organic solvent. However, it is also possible to use a
solvent during the blending process for more rapid or complete
blending, and then evaporate off the solvent when blending is
complete.
There is no critical upper limit on the amount of biologically
active substance incorporated except for that of an acceptable
solution or dispersion viscosity to maintain the physical
characteristics desired for the composition. The lower limit of the
substance incorporated into the delivery system is dependent upon
the activity of the drug and the length of time needed for
treatment. Thus, the amount of the biologically active substance
should not be so small that it fails to produce the desired
physiological effect, nor so large that the biologically active
substance is released in an uncontrollable manner.
Typically, within these limits, amounts of the biologically active
substance from about 1% up to about 65% can be incorporated into
the present delivery systems. However, lesser amounts may be used
to achieve efficacious levels of treatment for biologically active
substances that are particularly potent.
In addition, the polymer composition of the invention can also
comprise blends of the polymer of the invention with other
biocompatible polymers or copolymers, so long as the additional
polymers or copolymers do not interfere undesirably with the
biodegradable or mechanical characteristics of the composition.
Blends of the polymer of the invention with such other polymers may
offer even greater flexibility in designing the precise release
profile desired for targeted drug delivery or the precise rate of
biodegradability desired. Examples of such additional biocompatible
polymers include other poly(phosphoesters), poly(carbonates),
polytesters), poly(orthoesters), poly(phosphazenes), poly(amides),
poly(urethanes), poly(imino-carbonates), and poly(anhydrides).
Pharmaceutically acceptable polymeric carriers may also comprise a
wide range of additional materials. Without being limited thereto,
such materials may include diluents, binders and adhesives,
lubricants, disintegrants, colorants, bulking agents, flavorings,
sweeteners, and miscellaneous materials such as buffers and
adsorbents, in order to prepare a particular medicated composition,
with the condition that none of these additional materials will
interfere with the biocompatibility, biodegradability and
flowability or flexibility of the polymer compositions of the
invention.
For delivery of a biologically active substance, the biologically
active substance is added to the polymer composition. The
biologically active substance is either dissolved to form a
homogeneous solution of reasonably constant concentration in the
polymer composition, or dispersed to form a suspension or
dispersion within the polymer composition at a desired level of
"loading" (grams of biologically active substance per grams of
total composition including the biologically active substance,
usually expressed as a percentage).
While it is possible that the biodegradable polymer or the
biologically active agent may be dissolved in a small quantity of a
solvent that is non-toxic to more efficiently produce a
homogeneous, monolithic distribution or a fine dispersion of the
biologically active agent in the flexible or flowable composition,
it is an advantage of the invention that, in a preferred
embodiment, no solvent is needed to form a flowable composition.
Moreover, the use of solvents is preferably avoided because, once a
polymer composition containing solvent is placed totally or
partially within the body, the solvent dissipates or diffuses away
from the polymer and must be processed and eliminated by the body,
placing an extra burden on the body's clearance ability at a time
when illness or injury may have already deleteriously affected
it.
However, when a solvent is used to facilitate mixing or to maintain
the flowability of the polymer composition of the invention, it
should be non-toxic, otherwise biocompatible, and should be used in
minimal amounts. Solvents that are toxic clearly should not be used
in any material to be placed even partially within a living body.
Such a solvent also must not cause tissue irritation or necrosis at
the site of administration.
Examples of suitable biocompatible solvents, when used, include
N-methyl-2-pyrrolidone, 2-pyrrolidone, ethanol, propylene glycol,
acetone, methyl acetate, ethyl acetate, methyl ethyl ketone,
dimethylformamide, dimethyl sulfoxide, tetrahydrofuran,
caprolactam, dimethyl-sulfoxide, oleic acid, or
1-dodecylazacycloheptan-2-one. Preferred solvents include
N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, and
acetone because of their solvating ability and their
biocompatibility.
Flowable or Flexible Delivery Systems
In its simplest form, a biodegradable therapeutic agent delivery
system consists of a solution or dispersion of a biologically
active substance in a polymer matrix having an unstable
(biodegradable) bond incorporated into the polymer backbone.
Cleavage of the bond converts a water-insoluble polymer into
water-soluble, low molecular weight polymer fragments that can be
excreted from the body.
The biologically active substance is typically released from the
polymeric matrix at least as quickly as the matrix biodegrades in
vivo. With some biologically active substances, the substance will
be released only after the polymer has been degraded to a point
where a non-diffusing substance has been exposed to bodily fluids.
As the polymer begins to degrade, the biologically active substance
that was completely surrounded by the polymer matrix begins to be
liberated. However, with this mechanism, a long peptide chain that
is physically entangled in a rigid solid implant structure may tend
to degrade along with the matrix and break off from the remainder
of the peptide chain, thereby releasing incomplete fragments of
molecules.
With the polymer compositions of the invention, however, the
polymer will typically degrade after the peptide or protein has
been released in part. In a particularly preferred mechanism, when
a peptide chain is being released from the composition of the
invention, the composition remains flexible and allows a
large-molecule protein to, at least partially, diffuse through the
polymeric matrix prior to its own or the polymer's
biodegradation.
The initial release rate of proteins from the compositions is
therefore generally diffusion-controlled through channels in the
matrix structure, the rate of which is inversely proportional to
the molecular weight of the protein. Once polymer degradation
begins, however, the protein remaining in the matrix may also be
released by the forces of erosion.
The biodegradable amorphous matrices of the invention typically
contain polymer chains that are associated with other chains. These
associations can be created by a simple entanglement of polymer
chains within the matrix, as opposed to hydrogen bonding or Van der
Vaals interactions or between crystalline regions of the polymer or
interactions that are ionic in nature. Alternatively, the synthesis
of block copolymers or the blending of two different polymers can
be used to create viscous, "putty-like" materials with a wide
variation in physical and mechanical properties.
When the biologically active substance is a protein, interactions
between specific proteins and the polymeric materials often also
affect the characteristics of the composition. Important factors
include:
(i) the molecular weight of the protein, which is an important
parameter with regard to diffusion characteristics;
(ii) the isoelectric point of the protein, which governs
charge-charge interactions;
(iii) the presence of cysteines on the protein, which may
participate in the formation of intermolecular disulfide bonds;
(iv) the primary amino acid sequence of the protein, which may be
susceptible to chemical modification in association with a
polymeric material;
(v) the presence or absence of carbohydrates on the protein, which
may enhance or prevent interaction with polymeric materials;
(vi) the relative hydrophobicity of a protein, which can interact
with hydrophobic sites on a polymer; and
(vii) the heterogeneity of the protein, which often exists when
proteins are produced by recombinant methods.
In a particularly preferred embodiment, the composition of the
invention is sufficiently flowable to be injected into the body. It
is particularly important that the injected composition result in
minimal tissue irritation after injection or otherwise being placed
into direct contact with vasculated tissues.
The biologically active substance of the composition and the
polymer of the invention may form a homogeneous matrix, or the
biologically active substance may be encapsulated in some way
within the polymer. For example, the biologically active substance
may be first encapsulated in a microsphere and then combined with
the polymer in such a way that at least a portion of the
microsphere structure is maintained. Alternatively, the
biologically active substance may be sufficiently immiscible in the
polymer of the invention that it is dispersed as small droplets,
rather than being dissolved, in the polymer. Either form is
acceptable, but it is preferred that, regardless of the homogeneity
of the composition, a significant portion of the biologically
active substance is released in vivo prior to the biodegradation of
the polymer by hydrolysis of the phosphoester bond.
In one embodiment, the polymer composition of the invention is used
to form a soft, drug-delivery "depot" that can be administered as a
liquid, for example, by injection, but which remains sufficiently
viscous to maintain the drug within the localized area around the
injection site. The degradation time of the depot so formed can be
varied from several days to a few years, depending upon the polymer
selected and its molecular weight. By using a polymer composition
in flowable form, even the need to make an incision can be
eliminated. In any event, the flexible or flowable delivery "depot"
will adjust to the shape of the space it occupies within the body
with a minimum of trauma to surrounding tissues.
The flexible or flowable polymer composition of the invention can
be placed anywhere within the body, including soft tissue such as
muscle or fat; hard tissue such as bone or cartilage; a cavity such
as the periodontal, oral, vaginal, rectal or nasal cavity; or a
pocket such as a periodontal pocket or the cul-de-sac of the eye.
The composition may also be sprayed onto or poured into open wounds
or used as a site delivery system during surgery.
When flowable, the composition of the invention can be injected
into deeper wounds, such as burn wounds, to prevent the formation
of deep scars. The composition can also be used to act as a
temporary barrier in preventing the direct adhesion of different
types of tissue to each other, for example, after abdominal
surgery, due to its ability to encapsulate tissues, organs and
prosthetic devices.
In gene therapy, the flexible or flowable composition of the
invention may be useful for providing a means for delivering genes
to patients without involving a cell-based system. In particular,
the composition of the invention may be injected into sites that
would otherwise be inaccessible for direct delivery of gene
vectors. In addition, depending upon the need for continued gene
therapy, the sustained release capability of the biologically
active substance from the composition of the invention would
eliminate the need for repeated invasive procedures to re-introduce
the gene vector to the involved site.
In orthopedic applications, the flowable or flexible composition of
the invention may be useful for repairing bone defects and
connective tissue injuries. For example, the biodegradable
composition can be loaded with bone morphogenetic proteins to form
a bone graft useful for even large segmental defects, when the bone
can be immobilized and supported. The composition can also be
injected into an appropriate orthopedic space to facilitate cell
adhesion and proliferation before the polymeric matrix degrades to
non-toxic residues.
Once injected, the polymer composition of the invention should
remain in at least partial contact with a biological fluid, such as
blood, internal organ secretions, mucous membranes, cerebrospinal
fluid and the like. For drug-delivery systems, the implanted or
injected composition will release the biologically active substance
contained within its matrix at a controlled rate until the
substance is depleted, following the general rules for diffusion or
dissolution of a biologically active substance from a biodegradable
polymeric matrix.
The following examples are illustrative of preferred embodiments of
the invention and are not to be construed as limiting the invention
thereto. All polymer molecular weights are average molecular
weights. All percentages are based on the percent by weight of the
final delivery system or formulation being prepared, unless
otherwise indicated, and all totals equal 100% by weight.
EXAMPLES
Example 1
Synthesis of the Poly(phosphoester) P(trans-CHDM-HOP) ##STR9##
Under an argon stream, 10 g of trans-1,4-cyclohexane dimethanol
(CHDM), 1.794 g of 4-dimethylaminopyridine (DMAP), 15.25 ml (14.03
g) of N-methyl morpholine (NMM), and 50 ml of methylene chloride,
were transferred into a 250 ml flask equipped with a funnel. The
solution in the flask was cooled down to -15.degree. C. with
stirring, and a solution of 15.19 g of hexyl phosphorodichloridate
(HOP) in 30 ml of methylene chloride was added through the funnel.
The temperature of the reaction mixture was raised to the boiling
point gradually and maintained at reflux temperature overnight.
The reaction mixture was filtered, and the filtrate was evaporated
to dryness. The residue was re-dissolved in 100 ml of chloroform.
This solution was washed with 0.1 M solution of a mixture of HCl
and NaCl, dried over anhydrous Na.sub.2 SO.sub.4, and quenched into
500 ml of ether. The resulting flowable precipitate was collected
and dried under vacuum to form a clear pale yellow gel-like polymer
with the flow characteristics of a viscous syrup. The yield for
this polymer was 70-80%. The structure of P(trans-CHDM-HOP) was
ascertained by .sup.31 P-NMR and .sup.1 H-NMR spectra, as shown in
FIG. 1, and by FT-IR spectra. The molecular weights (Mw=8584;
Mn=3076) were determined by gel permeation chromatography (GPC), as
shown in FIG. 2, using polystyrene as a calibration standard.
Example 2
Synthesis of the Poly(Phosphoester) P(cis & trans-CHDM-HOP)
Poly(phosphoester) P(cis/trans-l1,4-cyclohexane-dimethanol hexyl
phosphate) was prepared by following the procedure described above
in Example 1 except that a mixture of cis- and
trans-1,4-cyclohexanedimethanol was used as the starting material.
As expected, the product cis-/trans-P(CHDM-HOP) was less viscous
than the transisomer obtained in Example 1.
Example 3
Synthesis of Low Molecular Weight P(CHDM-HOP)
Under an argon stream, 10 g of trans-1,4-cyclohexane dimethanol
(CHDM), 15.25 mL (14.03 g) of N-methyl morpholine (NMM), and 50 mL
of methylene chloride were transferred into a 250 mL flash equipped
with a funnel. The solution in the flask was cooled down to
-40.degree. C. with stirring. A solution of 15.19 g of hexyl
phosphoro dichloridate (HOP) in 20 mL of methylene chloride was
added through the funnel, and an additional 10 mL of methylene
chloride was used to flush through the funnel. The mixture was then
brought up to room temperature gradually and kept stirring for four
hours.
The reaction mixture was filtered, and the filtrate was evaporated
to dryness. The residue was re-dissolved in 100 ml of chloroform.
This solution was washed with 0.5 M mixture of HCl-NaCl solution,
washed with saturated NaCl solution, dried over anhydrous Na.sub.2
SO.sub.4, and quenched into a 1:5 ether-petroleum mixture. The
resulting oily precipitate was collected and dried under vacuum to
form a clear, pale yellow viscous material. The structure of the
product was confirmed by .sup.1 H-NMR, .sup.31 P-NMR and FT-IR
spectra.
Example 4
Synthesis of the Poly(phosphoester) P(trans-CHDM-BOP) ##STR10##
Under an argon stream, 10 g of trans-1,4-cyclohexane dimethanol
(CHDM), 0.424 g (5%) of 4-dimethylamino-pyridine (DMAP), 15.25 mL
(14.03 g) of N-methyl morpholine (NMM) and 50 mL of methylene
chloride were transferred into a 250 mL flask equipped with a
funnel. The solution in the flask was cooled down to -40.degree. C.
with stirring. A solution of 13.24 g of butyl
phosphoro-dichloridate (BOP) in 20 mL of methylene chloride was
added through the funnel, with an additional 10 mL of methylene
chloride being used to flush through the funnel. The mixture was
heated to the boiling point gradually, and kept refluxing for four
hours. The reaction mixture was filtered, and the filtrate was
evaporated to dryness, taking care to keep the temperature below
60.degree. C. The residue was redissolved in 100 mL of chloroform.
The solution formed was washed with 0.5 M of HCl-NaCl solution and
saturated NaCl solution, dried over anhydrous Na.sub.2 SO.sub.4,
and quenched into a 1:5 ether-petroleum mixture. The resulting oily
precipitate was collected and dried under vacuum to produce a
clear, pale yellow viscous material.
Example 5
Synthesis of the Poly(phosphoester) P(trans-CHDM-EOP) ##STR11##
The polymer p(CHDM-EOP) was prepared by the method of Example 1
using, as starting materials, trans-1,4-cyclohexane dimethanol
(CHDM) and ethyl phosphoro-dichloridate (EOP).
Example 6
Rheological Properties of P(trans-CHDM-HOP)
P(trans-CHDM-HOP) remained in a flowable gel-like state at room
temperature. The polymer exhibited a steady viscosity of
327Pa.multidot.s at 25.degree. C. (shown in FIG. 3B), and a flowing
active energy of 67.5 KJ/mol (shown in FIG. 3A).
Example 7
In Vitro Cytotoxicity of P(trans-CHDM-HOP)
Cover slips were coated with P(trans-CHDM-HOP) by a spin coating
method. The coated coverslips were then dried and sterilized by UV
irradiation overnight under a hood. A P(trans-CHDM-HOP)-coated
cover slip was placed at the bottom of each well of a 6-well plate.
5.times.10.sup.5 HEK293 (human embryonic kidney) cells were plated
into each well and cultured for 72 hours at 37.degree. C. The
resulting cell morphology was examined, using tissue culture
polystyrene (TCPS) as a positive control. The cells growing on the
P(CHDM-HOP) surface proliferated at a slightly slower rate.
However, the morphology of cells grown on the polymer surface was
similar to the morphology of cells grown on the TCPS surface. See
FIG. 4A for the morphology of HEK293 cells grown on the polymer
surface and FIG. 4B for the morphology of HEK293 cells grown on a
TCPS surface, both after 72 hours of incubation.
Example 8
In Vitro Degradation of P(CHDM-Alkyl Phosphates)
Each of the following poly(phosphate)s was prepared as described
above:
TABLE I Polymer Side Chain P(CHDM-HOP) --O-hexyl group P(CHDM-BOP)
--O-butyl group P(CHDM-EOP) --O-ethyl group
A sample of 50 mg of each polymer was incubated in 5 mL of 0.1 M,
pH 7.4 phosphate buffer saline (PBS) at 37.degree. C. At various
points in time, the supernatant was poured out, and the polymer
samples were washed three times with distilled water. The polymer
samples were then extracted with chloroform, and the chloroform
solution was evaporated to dryness. The residue was analyzed for
weight loss by comparing with the original 50 mg sample. FIG. 5
graphically represents the effect of the side chain structure on
the in vitro degradation rate of poly(phosphates) in PBS.
Example 9
In Vitro Release Profile of Protein by P(CHDM-HOP)
The polymer P(CHDM-HOP) was blended with the protein FITC-BSA
(bovine serum albumin, a protein, tagged with the fluorescent label
FITC; "FITC-BSA") at a 2:1 (w/w) ratio (33% loading). Measured
amounts (66 mg or 104 mg) of the polymer-protein blend were placed
into 10 ml of PBS (0.1M, pH 7.4), a phosphate buffer. At regular
intervals (roughly every day), the samples were centrifuged, the
supernatant buffer was removed and subjected to absorption
spectroscopy (501 nm), and fresh amounts of buffer were added to
the samples. The resulting release curve, plotting the cumulative
percentage of FITC-BSA released versus time, is graphically
represented in FIG. 6. The loading level of the protein in both
cases was 33 weight %.
Example 10
In Vitro Protein Release Profile At Various Loading Levels
FITC-BSA was blended with P(CHDM-HOP) at different loading levels
(1%, 10% and 30%) at room temperature until the mixture formed a
homogenous paste. 60 mg of the protein-loaded polymer paste was
placed in 6 mL of 0.1 M phosphate buffer and constantly shaken at
37.degree. C. At various time points, samples were centrifuged, and
the supernatant was replaced with fresh buffer. The released
FITC-BSA in the supernatant was measured by UV spectrophotometry at
501 nm. FIG. 7 graphically represents the in vitro release kinetics
of FITC-BSA as a function of loading level.
Example 11
Effect of Side Chain Structure on In Vitro Protein Release Kinetics
of FITC-BSA
The following three polymers were prepared as described above:
P(CHDM-EOP)
P(CHDM-BOP) and
P(CHDM-HOP)
FITC-BSA was blended with each polymer at a 10% loading level at
room temperature to form a homogenous paste. 60 mg of the
protein-loaded polymer paste was placed in 6 mL of 0.1 M phosphate
buffer with constant shaking at 37.degree. C. At various time
points, samples were centrifuged, and the supernatant was replaced
with fresh buffer. The released FITC-BSA in the supernatant was
measured by UV spectrophotometry at 501 nm. FIG. 8 graphically
represents the in vitro effect of side chain variations on the
protein release kinetics of FITC-BSA at 10% loading level.
Example 12
In Vitro Small Molecular Weight Drug Release from P(CHDM-HOP)
A P(CHDM-HOP) paste containing doxorubicin, cisplatin, or
5-fluorouracil, was prepared by blending 100 mg of P(CHDM-HOP) with
1 mg of the desired drug at room temperature, respectively. An
aliquot of 60 mg of the drug-loaded paste was placed in 6 mL of 0.1
M phosphate buffer at 37.degree. C. with constant shaking, with
three samples being done for each drug being tested. At various
time points, the supernatant was replaced with fresh buffer
solution. The levels of doxorubicin and 5-fluorouracil in the
supernatant were quantified by UV spectrophotometry at 484 nm and
280 nm, respectively. The cisplatin level was measured with an
atomic absorbance spectrophotometer. FIG. 9 shows the release of
these low molecular weight drugs from P(CHDM-HOP).
Example 13
In Vitro Simultaneous Release Profile of Doxorubicin and Cisplatin
from P(CHDM-HOP)
A paste was made by blending 300 mg of P(CHDM-HOP) with 6 mg of
doxorubicin and 6 mg of cisplatin at room temperature to form a
uniform dispersion. A sample of 100 mg of the paste was incubated
in 10 mL of phosphate buffer (pH 7.4) at 37.degree. C. with
shaking. At different time points, samples were centrifuged, 9 mL
of the supernatant were withdrawn and replaced with fresh buffer.
The withdrawn supernatant was assayed spectrophotometrically at 484
nm to determine the amount of doxorubicin released into the
withdrawn supernatant, and the cisplatin release was measured by
atomic absorbance spectrophotometer. FIG. 10 graphically represents
the simultaneous release of cisplatin and doxorubicin from
P(CHDM-HOP).
Example 14
In Vitro Interleukin-2 Release from P(CHDM-HOP)
A paste was prepared by blending, with a spatula, 330 mg of
P(CHDM-HOP) with 3 mg of IL-2 at room temperature to form a uniform
dispersion. A sample of 95 mg of the P(CHDM-HOP)/IL-2 paste was
placed in 5 mL of 0.1 M phosphate buffer (pH 7.4) at 37.degree. C.
At various time points, the sample was centrifuged and 4 mL of the
supernatant of 4 mL was withdrawn and replaced. the withdrawn
supernatant was assayed for IL-2 by use of CTLL-2 culture, as
described above. The cumulative percentage of IL-2 released was
calculated based on the initial amount of IL-2 blended into the
paste. At the last time point, there was IL-2 still left in the
sample. FIG. 11 graphically represents the cumulative percentage of
released IL-2 from the P(CHDM-HOP) matrix versus time in days.
Example 15
In Vitro Release of Interleukin-2 from P(CHDM-HOP) in Tissue
Culture
A paste was prepared by blending, with a spatula, lyophilized human
Interleukin-2 ("IL-2", 18.times.10.sup.6 IU) with 240 mg of
P(CHDM-HOP) at room temperature until homogeneous. Three 80 mg
samples of the P(CHDM-HOP)/IL-2 paste were incubated with 1.5 mL of
tissue culture (RPMI1640 Medium containing 10% FCS) at 37.degree.
C. with constant shaking. At various time points, the samples were
centrifuged, and the supernatant was withdrawn and replaced with
fresh medium. The amount of IL-2 in the withdrawn supernatant
samples was determined by an ELISA assay.
The amount of biologically active IL-2 released was assayed by the
following CTLL cell culture method: CTLL cells were plated in a
96-well plate at a density of 2.times.10.sup.4 cells per well and
incubated with an aliquot of the withdrawn supernatant. After two
days of incubation, the rate of cell growth was evaluated by WST-1
assay. A calibration curve was constructed in parallel for the
assay of IL-2 release from P(CHDM-HOP) in tissue culture medium.
FIG. 12 shows the calibration curves constructed by the sustained
release of IL-2. The complete data establish that more than 30% of
the bioactivity was retained at all points in time.
Example 17
In Vivo Release of Interleukin-2 from P(CHDM-HOP)
A sample of P(CHDM-HOP) was sterilized by .gamma.-irradiation at
2.5 MRads and aseptically blended with IL-2 in the same manner as
described above in Example 15. Six female Balb/c mice, 6-8 weeks of
age, were injected subcutaneously with 50 mg of the IL-2 polymer
paste sample containing 3.5.times.10.sup.5 IU of IL-2. Two
additional mice received the same dose of IL-2 as a bolus
injection, and two additional mice received blank P(CHDM-HOP)
injection as a control.
At various time points, 50 .mu.L of blood samples were collected
from the tail vein. Blood samples from each group were combined and
diluted with HBSS supplemented with 1% BSA. The serum was separated
and assayed for IL-2 as described above. Sustained release of IL-2
was attained in vivo, with detectable levels of IL-2 present in the
serum, for up to three weeks after injection of the
P(CHDM-HOP)/IL-2-loaded paste. In contrast, the IL-2 levels were
undetectable after 48 hours in the mice injected with the IL-2
bolus. FIG. 13 graphically compares the pharmacokinetics of IL-2
administered either as a bolus or dispersed in a P(CHDM-HOP)
matrix. FIG. 14 depicts the histological examination of a
subcutaneous injection site from this in vivo experiment.
Example 18
In Vivo Biocompatibility of P(trans-CHDM-HOP)
The polymer P(trans-CHDM-HOP) was synthesized as described in
Example 1. To facilitate injection, ethyl alcohol was added to the
polymer at levels of 10% and 20% by volume to reduce the viscosity.
Samples of 25 .mu.L of the polymer alone, 25 .mu.L of the polymer
containing 10% alcohol, and 25 .mu.L of the polymer containing 20%
alcohol, were injected into the back muscles of Sprague Dawley
rats. Tissues at the injection sites were harvested at either three
or thirteen days post-injection, processed for paraffin histology,
stained with heamatoxylln, eosin dye and analyzed. Medical-grade
silicon oil was injected into the control group rats.
Histological examination of the back muscle sections of the rats
injected with the polymer diluted with ethanol showed no acute
inflammatory response. The level of macrophage presence was
comparable to that of the control group, which had been injected
with medical-grade silicon oil, and neutrophils were not present in
any of the samples taken on either the third or thirteenth day.
Example 18
Drug Sensitivity in an In Vitro Tumor Model
In vitro studies were done on the melanoma cell line B16/F10 using,
as the drug, doxorubicin ("DOX"), cisplatin, or 5-fluorouracil
("5-FU"). The B16/F10 cells were cultivated in the presence of
different concentrations of DOX, cisplatin and 5-FU. According to
the data, DOX showed the strongest inhibitory effect on the cell
culture, even at 0.1 .mu.g/mL.
Example 19
Controlled Delivery of Interleukin-2 and Doxorubicin from
P(CHDM-HOP) in an In Vivo Tumor Model
Lyophilized interleukin-2 ("IL-2") was purchased from Chiron, mouse
Interferon-.gamma. ("mIFN-.gamma.") was obtained from Boehringer
Mannheim, and doxorubicin hydrochloride ("DOX") was obtained from
Sigma. C57BL/6 mice, 6-8 weeks of age, were obtained from Charles
River. The aggressive melanoma cell line B16/F10 was used to cause
tumors in the mice, and the cells were maintained by weekly
passages. The polymer P(CHDM-HOP) was synthesized as described in
Example 1.
Mice were randomly allocated into groups as shown below in TABLE
II. The day of tumor injection with cells of the melanoma cell line
was denoted as Day 0. Each mouse received a subcutaneous injection
of 50 .mu.l (10.sup.5) tumor cells in phosphate buffer saline (PBS)
in the left flank. On Day 3 or Day 7, the tumor-bearing mice were
selectively injected in the right flank with one of the following
formulations: (1) a bolus of IL-2, (2) a bolus of DOX, (3) a
polymer paste of IL-2, (4) a polymer paste of DOX, (5) a polymer
paste containing both IL-2 and DOX, or (6) a polymer paste
containing both IL-2 and mIFN-.gamma.. A control group and negative
control group received no further injections on Day 3 or Day 7.
The bolus preparation of either IL-2 or DOX was prepared by
dissolving an appropriate amount of IL-2 or DOX in 50 .mu.l of
isotonic solution just prior to the injection. The polymer paste
formulations of either IL-2, DOX, a mixture of both IL-2 and DOX,
or a mixture of IL-2 and mIFN-.gamma., were prepared by blending 50
.mu.l of sterilized P(CHDM-HOP) with the drug(s) until
homogeneous.
TABLE II Allocation of Groups of Mice for In Vivo Tumor Model Day
of Number Injec- Group of Mice tion Formulation Control 5 --
Nothing Negative 5 -- Nothing Control Bolus IL-2 8 3 0.8 .times.
10.sup.6 IU Bolus DOX 8 3 0.5 mg Bolus DOX 8 7 0.5 mg Paste IL-2 10
3 0.8 .times. 10.sup.6 IU Paste IL-2 10 7 0.8 .times. 10.sup.6 IU
Paste DOX 10 3 0.5 mg Paste DOX 10 7 0.5 mg Paste (IL-2 + 10 3 0.8
.times. 10.sup.6 IU + DOX) 0.5 mg Paste (IL-2 + 10 7 0.8 .times.
10.sup.6 IU + DOX) 0.5 mg Paste (IL-2 + 10 3 10.sup.6 IU
mIFN-.gamma.)
On Day 28 and Day 42 of tumor growth, the tumor sizes of the
various mice were measured. The results are shown below in Table
III, which shows the numerical data for the growth of tumor volumes
on Day 28 and Day 42 and the number of mice who survived the
experiment per drug grouping. Tumor volume was calculated as half
the product of the length and the square of the width, in
accordance with the procedure of Osieka et al., 1981.
TABLE III CHDM-HOP Polymer as Carrier for Cytokine and Drug
Delivery in Melanoma Model Tumor Volume (mm.sup.3 .+-. SEM*) After
Initial Tumor Injection Number 28 days 42 days Group of Mice Number
of Mice Survived Control 5 No tumor No tumor Negative 5 2458 .+-.
1070.7 5656 Control 4 1 Bolus IL-2 8 1946 .+-. 505.6 3282 .+-.
1403.3 (3d) 8 4 Bolus Dox 8 1218.9 .+-. 304.1 3942.5 .+-. 1818 (3d)
8 5 Bolus Dox 8 1661.2 .+-. 301.8 4394.3 .+-. 741.3 (7d) 8 3 Paste
IL-2 10 934.1 .+-. 230 3183 .+-. 1223.4 (3d) 10 5 Paste IL-2 10
2709.8 .+-. 397.3 10491 .+-. 2485.5 (7d) 10 3 Paste Dox 10 1410
.+-. 475.3 4648.9 .+-. 1202.2 (3d) 8 7 Paste Dox 10 1480 .+-. 287
3915 .+-. 1739.7 (7d) 9 4 Paste (IL-2 + 10 657.3 .+-. 248.9 3362.8
.+-. 1120.1 DOX) (3d) 8 7 Paste (IL-2 + 10 857.2 .+-. 243.6 3449.8
.+-. 1285.9 DOX) (7d) 8 5 Paste (IL-2 + 10 1217.9 .+-. 168.4 4469.8
.+-. 2018.7 mIFN-.gamma.) (3d) 9 4 *Standard Error of the Mean
Based on these measurements, the distribution of tumors sizes were
graphically represented in FIG. 15 for Day 28 (four weeks after
tumor implantation) and in FIG. 16 for Day 42 (six weeks after
tumor implantation). The graphs were subdivided into plots
according to the different treatments given to the tumor-bearing
mice.
The results on Day 28 showed that, in comparison with the control
group (tumor without treatment) and the bolus injection of IL-2,
the group of mice receiving a polymer/IL-2 paste injection
successfully delayed the tumor's growth. However, for the group of
mice not receiving a polymer/IL-2 paste injection until Day 7, the
tumor had already become of substantial size by Day 7 and,
accordingly, a significant reduction in tumor size was not
observed.
Excellent tumor reduction was obtained with the combination of IL-2
and DOX. The average size of a tumor treated with an injection of a
polymer paste containing both IL-2 and DOX was significantly
smaller than the tumor in the control group. Specifically, the
average tumor size for mice receiving the IL-2 and DOX/polymer
paste on Day 3 was 657.3 mm.sup.3 as opposed to 2458 mm.sup.3 for
the control group. Even when treatment was delayed until Day 7 of
tumor growth, a therapeutic effect could still be seen with the
polymer paste formulation containing both IL-2 and DOX.
The results on Day 42 of tumor growth also confirmed that the Day 3
injection of polymer paste containing both IL-2 and DOX gave the
best result in delaying tumor growth. The combined therapy of IL-2
and DOX in a polymer paste of the invention resulted in the
occurrence of smaller sized tumors in more of the test animals.
According to the distribution data shown in FIG. 15, there were
four mice bearing tumors of less than 1000 mm.sup.3 in the case of
the combined IL-2 and DOX polymer paste therapy, as compared with
only one mouse inside that range for the polymer paste injection of
DOX alone. It was also clear that IL-2 alone did not provide the
most desirable effect, as evaluated on the 42nd day of tumor
growth. Despite the good distribution of small tumor sizes on the
28th day, the long-time survival data appeared to be adversely
affected, not only by the progression of tumor growth at that
point, but also by the lack of continued, controlled delivery of
IL-2 over a longer time period. With the polymer paste formulation
of both IL-2 and DOX, the polymer degraded slowly, allowing a
gradual decrease in the diffusion rate of the therapeutic agent
over time.
However, because of the significant difference of the distribution
in tumor sizes inside each group, the average tumor size as seen in
TABLE III did not provide a complete picture. A fuller appreciation
of the significance of the treatments of the invention can be
gained by comparing data from the distribution graph of FIG. 16,
which shows the dispersity in tumor sizes six weeks after tumor
implantation, with the survival curve shown in FIG. 17, which shows
the massive death of mice in all groups before the Day 42
measurement, except for the groups of animals that had received the
3rd day injection of paste containing either DOX alone or the
combination of IL-2 and DOX. Thus, the data, taken as a whole,
shows that the combined therapy of IL-2 and DOX in the paste both
significantly delayed tumor growth and extended life.
Early deaths about 3-4 days after the injections of the
DOX-containing polymer paste were thought to be due, at least in
part, to the toxic effect of DOX causing the deaths of the weaker
animals. Corresponding injections of bolus DOX did not produce
early death, probably because of the rapid distribution and
clearance from the body of the bolus-injected DOX.
Example 20
Incorporating Paclitaxel into P(CHDM-HOP) or P(CHDM-EOP)
100 mg of each of the polymers of Example 1, p(CHDM-HOP), and
Example 5, p(CHDM-EOP), was dissolved in ethanol at a concentration
of about 50%. After the polymer was completely dissolved, 5 mg of
paclitaxel powder (a chemotherapeutic drug) was added to the
solution and stirred until the powder was completely dissolved.
This solution was then poured into ice water to precipitate the
polymer composition. The resulting suspension was centrifuged,
decanted, and lyophilized overnight, to obtain a viscous gel-like
product.
Example 21
In Vitro Release of Paclitaxel from P(CHDM-HOP) and P(CHDM-EOP)
In a 1.7 mL plastic micro centrifuge tube, 5 mg of both of the
paclitaxel polymer formulations of Example 20 to be tested was
incubated with 1 mL of a buffer mixture of 80% PBS and 20% PEG 400
at 37.degree. C. Four samples of each formulation to be tested were
prepared. At specific time points, approximately every day, the
PBS:PEG buffer was poured off for paclitaxel analysis by HPLC, and
fresh buffer was added to the microcentrifuge tube. The release
study was terminated at day 26, at which point the remaining
paclitaxel in the polymer was extracted with a solvent to do a mass
balance on paclitaxel.
The resulting release curves for the release of paclitaxel from
both polymers are shown in FIG. 18. The total paclitaxel recovery
was 65% for the P(CHDM-HOP) formulation and 75% for the P(CHDM-EOP)
formulation.
Example 22
Preparation of P(CHDM-HOP)/Lidocaine Paste
A paste of P(CHDM-HOP) and lidocaine (base; Sigma, Cat. # L-7757)
was prepared by mechanically mixing as follows: 60 mg of
P(CHDM-HOP) and 16 mg of lidocaine were weighed onto a glass
microscope slide. The polymer and the lidocaine drug were
thoroughly mixed with a spatula until a uniform mixture was
obtained. The resulting lidocaine/polymer mixture formed a 24% w/w
lidocaine paste with the lidocaine remaining as a solid.
Example 23
In Vitro Release of Lidocaine from P(CHDM-HOP)
Approximately 10 mg of the lidocaine/polymer mixture prepared above
in Example 22 was placed in 2.0 mL of phosphate buffered solution
(PBS) (0.1 M, pH 7.4) at 37.degree. C. on a shaker. The buffer was
replaced at specific time points, and samples were withdrawn. The
lidocaine released from the polymer into the samples was assayed by
HLPC.
The results of three different samples of the lidocaine/polymer
mixture are graphically represented in FIG. 19. FIG. 20A displays
the cumulative amount of lidocaine released as a function of
incubation time and FIG. 20B shows the cumulative amount of
lidocaine released over the square root of time, demonstrating that
approximately 90% of the drug was released within one week. The
linear relationship between the amount of lidocaine released and
the square root of time indicated that the mechanism of drug
release was mainly through diffusion during the test period.
Example 24
Release of Lidocaine from P(CHDM-HOP)in a Rat Sciatic Nerve Model
In Vivo
Single jugular catheters were inserted into Male Sprague-Dawley
rats, approximately 150-200 g in weight. The rats were anesthetized
by i.p. injection with about 0.3-0.4 mL of an anesthetic cocktail
(25 mg/mL ketamine, 2.5 mg/mL xylazine and 14.5% 200 proof
ethanol). The sciatic nerve of the animal was identified. Each
animal received a single injection of either 25 mg or 50 mg of
lidocaine in either P(CHDM-HOP) or as a saline solution into its
sciatic nerve to block the nerve. Control group rats received an
equivalent amount of blank polymer injected into their sciatic
nerves.
The rats were observed over time, and scores were assigned to both
motor and nociceptive responses as follows:
Motor response and function
normal motor function=0,
slight foot drag=1,
moderate foot drag=2, and
no motor function=3;
Nociceltive response and function
normal nociceptive response=0,
slightly delayed nociceptive response=1,
delayed nociceptive response =2, and
no nociceptive response =3.
Blood samples were also collected at specific time points, and the
plasma concentration of lidocaine was assayed by HLPC.
FIG. 22 shows the plot of percent of maximum motor function effect
versus time after injection with 25 mg of lidocaine in P(CHDM-HOP)
or in saline solution. A maximum percentage effect of 100% on this
graph represents a score of "3" for "no motor response." All of the
rats injected with lidocaine-containing preparations exhibited
complete motor block during the first hour following injection.
Table IV below summarizes the duration of the lidocaine blocking
effects following injection of lidocaine in saline solution or in
P(CHDM-HOP).
TABLE IV Duration of Lidocaine Reaction Following Injection of
Lidocaine in Saline Solution or P(CHDM-HOP) Sensory Function Motor
Function Lidocaine Complete Partial Complete Partial Formulation
Block Block Block Block Blank P(CHDM-HOP) 0 0 0 0 25 mg Saline 2
hrs 48 hrs 1 hr 27 hrs solution 25 mg in 54 hrs 198 hrs 1 hr 198
hrs P(CHDM-HOP) 50 mg in 119 hrs 265 hrs .sup. 2 hrs 240 hrs
P(CHDM-HOP)
The duration of motor function blockage from the lidocaine in
P(CHDM-HOP) was clearly longer than that achieved by the lidocaine
saline solution. However, the extent of motor function blockage was
only partial, in that a rat could still move its leg with a slight
drag. It was also noted that the increase in complete motor
blockage was minimal even at the higher lidocaine concentration of
50 mg of lidocaine.
Table V below shows the percentage of rats exhibiting complete
blockage of the nociceptive response following the administration
of 25 mg of lidocaine either as a saline solution or in
P(CHDM-HOP).
TABLE V Percentage of Rats with Complete Nociceptive Response
Percentage of Rats with Complete Block of Nociceptive Response 25
mg Lidocaine 25 mg Lidocaine Time in P(CHDM-HOP) in Saline Solution
0.5 hrs 100 100 3 hrs 100 78 6 hrs 100 50 24 hrs 100 0 30 hrs 78 0
48 hrs 100 0 51 hrs 100 0 54 hrs 100 0 72 hrs 78 0 99 hrs 78 0 119
hrs 50 0 125 hrs 78 0 143 hrs 50 0 149 hrs 50 0
Compared with the lidocaine/saline solution, the
lidocaine/P(CHDM-HOP) formulation prolonged the sensory blocking
effect of lidocaine significantly.
FIG. 21 plots the percentage of maximum nociceptive effect versus
time after injection with 25 mg of lidocaine in either P(CHDM-HOP)
or saline solution. The maximum percentage effect of 100% on this
graph represented a score of "3", i.e., "no nociceptive response."
Again, compared with the data from the lidocaine in saline
solution, a significantly prolonged local anesthetic effect was
observed in the lidocaine/P(CHDM-HOP) group.
It was noted that recovery from the motor block occurred well
before complete recovery from the sensory block in both the
lidocaine/saline solution and the lidocaine/P(CHDM-HOP)
formulations. The rats could often move around with their hind limb
and still exhibit no apparent response to pain stimuli. Because
complete responsiveness to nociception was recovered well after the
recovery of motor function, pharmaceutical compositions of the
invention are believed to be well-suited for the clinical
administration of local anesthetics and the management of chronic
pain.
FIG. 23 shows the lidocaine concentration in plasma following
injection of 25 mg of lidocaine in saline solution, 25 mg of
lidocaine in P(CHDM-HOP), and 50 mg of lidocaine in P(CHDM-HOP). By
increasing the concentration of lidocaine in the polymer
formulation, the duration of the anesthetic function was extended
with a minimal increase in the lidocaine concentration in systemic
circulation, indicating that diffusion of the majority of the drug
was restricted to the local area.
The invention being thus described, it will be obvious that the
same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of at the
invention, and all such modifications are intended to be a included
within the scope of the following claims.
* * * * *